Factor viii complex with xten and von willebrand factor protein, and uses thereof

ABSTRACT

The present invention provides a chimeric protein comprising a VWF protein comprising the D′ domain and D3 domain of VWF, one or more XTEN sequence, and a FVIII protein, wherein the VWF fragment, the XTEN sequence, or the FVIII protein are linked to or associated with each other. The chimeric protein can further comprise one or more Ig constant region or a portion thereof (e.g., an Fc region). A polypeptide chain comprising a VWF fragment of the invention binds to or is associated with a polypeptide chain comprising a FVIII protein linked to an XTEN sequence and the polypeptide chain comprising the VWF fragment can prevent or inhibit binding of endogenous VWF to the FVIII protein linked to the XTEN sequence. By preventing or inhibiting binding of endogenous VWF to the FVIII protein, which is a half-life limiting factor for FVIII, the VWF fragment can induce extension of half-life of the chimeric protein comprising a FVIII protein. The invention also includes nucleotides, vectors, host cells, methods of using the VWF fragment, or the chimeric proteins.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.16/154,310, filed Oct. 8, 2018, which is a division of U.S. patentapplication Ser. No. 14/413,765, filed Jan. 9, 2015, now U.S. Pat. No.10,138,291, which is a 35 U.S.C. § 371 filing of International PatentApplication No. PCT/US2013/049989, filed Jul. 10, 2013, which claimspriority to U.S. Provisional Patent Application Nos. 61/840,811, filedJun. 28, 2013; 61/827,158, filed May 24, 2013; 61/801,504, filed Mar.15, 2013; 61/801,544, filed Mar. 15, 2013; 61/759,819, filed Feb. 1,2013; and 61/670,401, filed Jul. 11, 2012, the entire disclosures ofwhich are hereby incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The instant application contains a Sequence Listing, which has beensubmitted in ASCII format via EFS-Web, and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 23, 2021, isnamed “719865_SA9-428USDIV2_ST25.txt” and is 577,268 bytes in size.

BACKGROUND OF THE INVENTION

Haemophilia A is a bleeding disorder caused by defects in the geneencoding coagulation factor VIII (FVIII) and affects 1-2 in 10,000 malebirths. Graw et al., Nat. Rev. Genet. 6(6): 488-501 (2005). Patientsaffected with hemophilia A can be treated with infusion of purified orrecombinantly produced FVIII. All commercially available FVIII products,however, are known to have a half-life of about 8-12 hours, requiringfrequent intravenous administration to the patients. See Weiner M. A.and Cairo, M. S., Pediatric Hematology Secrets, Lee, M. T., 12.Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D.Thromb. Res. 122 Suppl 4:S2-8 (2008). In addition, a number ofapproaches have been tried in order to extend the FVIII half-life. Forexample, the approaches in development to extend the half-life ofclotting factors include pegylation, glycopegylation, and conjugationwith albumin. See Dumont et al., Blood. 119(13): 3024-3030 (Publishedonline Jan. 13, 2012). Regardless of the protein engineering used,however, the long acting FVIII products currently under development arereported to have limited half-lives—only to about 1.5 to 2 hours inpreclinical animal models. See id. Consistent results have beendemonstrated in humans, for example, rFVIIIFc was reported to improvehalf-life up to ˜1.7 fold compared with ADVATE® in hemophilia Apatients. See Id. Therefore, the half-life increases, despite minorimprovements, may indicate the presence of other T1/2 limiting factors.See Liu, T. et al., 2007 ISTH meeting, abstract #P-M-035; Henrik, A. etal., 2011 ISTH meeting, abstract #P=MO-181; Liu, T. et al., 2011 ISTHmeeting abstract #P-WE-131.

Plasma von Willebrand Factor (VWF) has a half-life of approximately 12hours (ranging from 9 to 15 hours).http://www.nhlbi.nih.gov/guidelines/vwd/2_scientificoverview.htm (lastvisited Oct. 22, 2011). The VWF half-life may be affected by a number offactors: glycosylation pattern, ADAMTS-13 (a disintegrin andmetalloprotease with thrombospondin motif-13), and various mutations inVWF.

In plasma, 95-98% of FVIII circulates in a tight non-covalent complexwith full-length VWF. The formation of this complex is important for themaintenance of appropriate plasma levels of FVIII in vivo. Lenting etal., Blood 92(11): 3983-96 (1998); Lenting et al., J. Thromb. Haemost.5(7): 1353-60 (2007). The full-length wild-type FVIII is mostly presentas a heterodimer having a heavy chain (MW 200 kD) and a light chain (MW73 kD). When FVIII is activated due to proteolysis at positions 372 and740 in the heavy chain and at position 1689 in the light chain, the VWFbound to FVIII is removed from the activated FVIII. The activated FVIII,together with activated factor IX, calcium, and phospholipid (“tenasecomplex”), induces the activation of factor X, generating large amountsof thrombin. Thrombin, in turn, then cleaves fibrinogen to form solublefibrin monomers, which then spontaneously polymerize to form the solublefibrin polymer. Thrombin also activates factor XIII, which, togetherwith calcium, serves to crosslink and stabilize the soluble fibrinpolymer, forming crosslinked (insoluble) fibrin. The activated FVIII iscleared fast from the circulation by proteolysis.

Due to the frequent dosing and inconvenience caused by the dosingschedule, there is still a need to develop FVIII products requiring lessfrequent administration, i.e., a FVIII product that has a half-lifelonger than the 1.5 to 2 fold half-life limitation.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a chimeric protein comprising (i) avon Willebrand Factor (VWF) fragment comprising the D′ domain and the D3domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, whereinthe VWF fragment and the XTEN sequence are linked by an optional linkerand wherein the VWF fragment or the XTEN sequence is linked to orassociated with the FVIII protein. The chimeric protein can comprise asingle polypeptide chain comprising the VWF fragment, the XTEN sequence,and the FVIII protein, or two polypeptide chains, a first chaincomprising the VWF fragment and the second chain comprising the FVIIIprotein, wherein the XTEN polypeptide is linked either to the VWFfragment or the FVIII protein.

In one embodiment, the chimeric protein of the invention comprises aformula comprising:

V-X-FVIII,  (a)

FVIII-X-V,  (b)

V-X:FVIII,  (c)

X-V:FVIII,  (d)

FVIII:V-X, or  (e)

FVIII:X-V,  (f)

wherein V comprises a VWF fragment,X comprises one or more XTEN sequences, andFVIII comprises a FVIII protein. The hyphen (-) can be a peptide bond ora linker, e.g., a cleavable linker, while the colon (:) represents achemical association or a physical association between the polypeptides,for example a covalent or non-covalent bond.

In another embodiment, the chimeric protein further comprises (iv) animmunoglobulin (Ig) constant region or a portion thereof (also indicatedas F1 or a first Ig constant region or a portion thereof) linked to theVWF fragment, the XTEN sequence, the FVIII protein, or any combinationsthereof. In other embodiments, the chimeric protein further comprises anadditional Ig constant region or a portion thereof (also indicated as F2or a second Ig constant region or a portion thereof). The first Igconstant region or a portion thereof can be linked to the VWF fragmentor the XTEN sequence, and the second Ig constant region can be linked tothe FVIII protein. The first Ig constant region, the second Ig constantregion or a portion thereof, or both can extend the half-life of theFVIII protein.

In some embodiments, the second Ig constant region or a portion thereof(F2) is linked to the VWF fragment by a linker, e.g., a processablelinker. In other embodiments, the second Ig constant region or a portionthereof (F2) is associated with the (first) Ig constant region or aportion thereof (F1). The second Ig constant region or a portion thereof(F2) and the first Ig constant region or a portion thereof (F1) can beidentical or different. The second Ig constant region or a portionthereof can be associated with the Ig constant region or a portionthereof by a covalent bond, e.g., a disulfide bond. The VWF fragmentlinked to the first Ig constant region or a portion thereof may also beassociated with the FVIII protein linked to the second Fc region by anon-covalent bond. In certain embodiments, the FVIII protein can furthercomprise one or more additional XTEN sequences which are linked to theC-terminus or N-terminus of the FVIII protein or inserted immediatelydownstream of one or more amino acids in the FVIII protein (e.g., one ormore XTEN insertion sites). In some embodiments, the half-life of theFVIII protein is extended, compared to wild type FVIII or a FVIIIprotein without the VWF fragment.

In some embodiments, the chimeric protein comprises a formulacomprising:

V-L2-X-L1-F1:FVIII-L3-F2;  (g)

V-L2-X-L1-F1:F2-L3-FVIII;  (h)

F1-L1-X-L2-V:FVIII-L3-F2;  (i)

F1-L1-X-L2-V:F2-L3-FVIII;  (j)

V-L2-X-L1-F1-L4-FVIII-L3-F2;  (k)

F2-L3-FVIII-L4-F1-L1-X-L2-V;  (l)

FVIII-L3-F2-L4-V-L2-X-L1-F1; or  (m)

F1-L1-X-L2-V-L4-F2-L3-FVIII,  (n)

wherein V comprises a VWF fragment,each of L1, L2, and L3 comprises an optional linker, e.g., a cleavablelinker,L4 is an optional linker, e.g., a processable linker,FVIII comprises a FVIII protein,X comprises one or more XTEN sequences,F1 comprises an optional first Ig constant region or a portion thereof,F2 comprises an optional second Ig constant region or a portion thereof,and(:) is a covalent bond or non-covalent bond.

The present invention is also directed to a chimeric protein comprising(i) a FVIII protein, (ii) an XTEN sequence, and (iii) an Ig constantregion or a portion thereof, wherein the XTEN sequence is linked to theFVIII protein by an optional linker at the N-terminus or C terminus ofthe FVIII protein or inserted immediately downstream of one or moreamino acids in the FVIII protein (e.g., one or more insertion sites) andwherein the Ig constant region or a portion thereof is linked to orassociated with the FVIII protein or the XTEN sequence. In oneembodiment, the Ig constant region or a portion thereof useful for thechimeric protein comprises a first Fc region. In another embodiment, thechimeric protein further comprises an additional Ig constant region or aportion thereof. The additional Ig constant region or a portion thereofuseful for the invention can comprise a second Fc region, which islinked to or associated with the first Fc region, e.g., by a covalentbond. In other embodiments, the first Fc region is linked to the secondFc region by a linker, e.g., a processable linker.

In other aspects, a chimeric protein comprises (i) a FVIII protein, (ii)an XTEN sequence, (iii) a VWF fragment, and (iv) an Ig constant regionor a portion thereof, which comprises the D′ domain and the D3 domain ofVWF, wherein the XTEN sequence is linked to the FVIII protein by anoptional linker at the N-terminus or C terminus of the FVIII protein orinserted immediately downstream of one or more amino acids in the FVIIIprotein (e.g., one or more insertion sites), the VWF fragment is linkedto or associated with the FVIII protein or the XTEN sequence, and the Igconstant region or a portion thereof is linked to the FVIII protein, theXTEN sequence, the VWF fragment, or any combinations thereof.Non-limiting examples of the chimeric proteins may comprise a formula,which comprises:

FVIII(X1)-L1-F1:V-L2-X2-L3-F2;  (1)

FVIII(X1)-L1-F1:F2-L3-X2-L2-V;  (2)

F1-L1-FVIII(X1):V-L2-X2-L3-F2;  (3)

F1-L1-FVIII(X1):F2-L3-X2-L2-V;  (4)

FVIII(X1)-L1-F1-L4-V-L2-X2-L3-F2;  (5)

FVIII(X1)-L1-F1-L4-F2-L3-X2-L2-V;  (6)

F1-L1-FVIII(X1)-L4-V-L2-X2-L3-F2, or  (7)

F1-L1-FVIII(X1)-L4-F2-L3-X2-L2-V,  (8)

wherein FVIII(X1) comprises a FVIII protein and one or more XTENsequences, wherein one or more of the XTEN sequences are linked to theN-terminus or C-terminus of the FVIII protein or inserted immediatelydownstream of one or more amino acids in the FVIII protein (e.g., one ormore XTEN insertion sites);each of L1, L2, or L3 comprises an optional linker, e.g., a cleavablelinker;L4 is a linker, a processable linker;X2 comprises one or more XTEN sequences;F1 comprises an Ig constant region or a portion thereof;F2 comprises an optional additional Ig constant region or a portionthereof, andV comprises a VWF fragment;(-) is a peptide bond or one or more amino acids; and(:) comprises a covalent bond or a non-covalent bond.

One aspect of the invention is that the VWF fragment useful for thechimeric protein does not bind to a VWF clearance receptor, whichprevents or inhibits interaction of the FVIII protein with endogenousVWF. The chimeric protein comprising the VWF fragment thus has reducedclearance or is not cleared through a VWF clearance pathway. Anotheraspect of the invention is that the VWF fragment is capable ofprotecting the FVIII protein from one or more protease cleavages,protecting the FVIII protein from activation, stabilizing the heavychain and/or the light chain of the FVIII protein, or preventingclearance of the FVIII protein by one or more scavenger receptors.

Because of the VWF fragment's ability to prevent or inhibit interactionbetween the FVIII protein and endogenous VWF, the half-life of the FVIIIprotein, is extended compared to a FVIII protein without the VWFfragment. In one embodiment, the half-life of the FVIII protein isextended at least about 1.5 times, at least about 2 times, at leastabout 2.5 times, at least about 3 times, at least about 4 times, atleast about 5 times, at least about 6 times, at least about 7 times, atleast about 8 times, at least about 9 times, at least about 10 times, atleast about 11 times, or at least about 12 times longer than wild typeFVIII. In another embodiment, the half-life of the FVIII protein is atleast about 10 hours, at least about 11 hours, at least about 12 hours,at least about 13 hours, at least about 14 hours, at least about 15hours, at least about 16 hours, at least about 17 hours, at least about18 hours, at least about 19 hours, at least about 20 hours, at leastabout 21 hours, at least about 22 hours, at least about 23 hours, atleast about 24 hours, at least about 36 hours, at least about 48 hours,at least about 60 hours, at least about 72 hours, at least about 84hours, at least about 96 hours, or at least about 108 hours.

The Ig constant region or a portion thereof useful for the chimericprotein comprises a first Fc region, which is linked to the VWF fragmentby an optional linker, e.g., a cleavable linker. The chimeric proteincan further comprise an additional Ig constant region or a portionthereof, which is linked to the FVIII protein or the XTEN sequence, theIg constant region or a portion thereof, the VWF fragment, or anycombinations thereof by an optional linker. In one embodiment, theadditional Ig constant region or a portion thereof is linked to theFVIII protein by an optional linker. The additional Ig constant regionor a portion thereof can comprise a second Fc region.

The Ig constant region or a portion thereof useful in the presentinvention and the additional Ig constant region or a portion thereofuseful in the present invention are identical or different.

In some aspects, the FVIII protein is linked to an XTEN sequence at theC-terminus or the N-terminus of the FVIII protein or insertedimmediately downstream of one or more amino acids in mature native humanFVIII (e.g., one or more insertion sites) or any combinations thereof.One or more insertion sites in the FVIII protein can be located withinone or more domains of the FVIII protein selected from the groupconsisting of the A1 domain, the a1 acidic region, the A2 domain, the a2acidic region, the A3 domain, the B domain, the C1 domain, the C2domain, and any combinations thereof or between one or more domains ofthe FVIII protein selected from the group consisting of the A1 domainand a1 acidic region, the a1 acidic region and A2 domain, the A2 domainand a2 acidic region, the a2 acidic region and B domain, the B domainand A3 domain, the A3 domain and C1 domain, the C1 domain and C2 domain,and any combinations thereof or between two domains of the FVIII proteinselected from the group consisting of the A1 domain and a1 acidicregion, the a1 acidic region and A2 domain, the A2 domain and a2 acidicregion, the a2 acidic region and B domain, the B domain and A3 domain,the A3 domain and C1 domain, the C1 domain and C2 domain, and anycombinations thereof.

In one embodiment, the one or more insertion sites are locatedimmediately downstream of one or more amino acids in mature native humanFVIII (e.g., SEQ ID NO: 4 [mature FVIII sequence-full length]) selectedfrom the group consisting of the amino acid residues in Table 7, 8, 9,10, 11, or any combinations thereof.

In another embodiment, the one or more insertion sites are located inone or more permissive loops of mature native human FVIII. In otherembodiments, the one or more insertion sites are located in the a3region of mature native human FVIII. For example, an XTEN sequence canbe inserted immediately downstream of amino acid 1656 corresponding toSEQ ID NO: 4 (full length mature FVIII). In other embodiments, a FVIIIprotein is linked to at least two XTEN sequences, a first XTEN sequenceinserted within the a3 region, and a second XTEN sequence insertedwithin a permissive loop in the FVIII protein (e.g., A1-1, A1-2, A2-1,A2-2, A3-1, or A3-2). In still other embodiments, a FVIII protein islinked to at least three XTEN sequences, a first XTEN sequence insertedwithin the a3 region and a second XTEN sequence and a third XTENsequence inserted within one or two permissive loop in the FVIII protein(e.g., A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2).

In certain embodiments, the one or more insertion sites for one or moreXTEN insertions are immediately downstream of one or more amino acids(numbered relative to mature FVIII sequence) selected from the groupconsisting of:

  (1) amino acid 3,   (2) amino acid 18, (3) amino acid 22, (4) aminoacid 26, (5) amino acid 32, (6) amino acid 40, (7) amino acid 60, (8)amino acid 65, (9) amino acid 81, (10) amino acid 116, (11) amino acid119, (12) amino acid 130, (13) amino acid 188, (14) amino acid 211, (15)amino acid 216, (16) amino acid 220, (17) amino acid 224, (18) aminoacid 230, (19) amino acid 333, (20) amino acid 336, (21) amino acid 339,(22) amino acid 375, (23) amino acid 399, (24) amino acid 403, (25)amino acid 409, (26) amino acid 416, (26) amino acid 442, (28) aminoacid 487, (29) amino acid 490, (30) amino acid 494, (31) amino acid 500,(32) amino acid 518, (33) amino acid 599, (34) amino acid 603, (35)amino acid 713, (36) amino acid 745, (37) amino acid 1656, (38) aminoacid 1711, (39) amino acid 1720, (40) amino acid 1725, (41) amino acid1749, (42) amino acid 1796, (43) amino acid 1802, (44) amino acid 1827,(45) amino acid 1861, (46) amino acid 1896, (47) amino acid 1900, (48)amino acid 1904, (49) amino acid 1905, (50) amino acid 1910, (51) aminoacid 1937, (52) amino acid 2019, (53) amino acid 2068, (54) amino acid2111, (55) amino acid 2120, (56) amino acid 2171, (57) amino acid 2188,(58) amino acid 2227, (59) amino acid 2277, and (60) two or morecombinations thereof.

In some embodiments, one XTEN is inserted in the FVIII protein. In someembodiments, two XTENs are inserted in the FVIII protein. In someembodiments, 3 XTENs are inserted in the FVIII protein.

In a particular example, a first XTEN is inserted immediately downstreamof amino acid 26 corresponding to SEQ ID NO: 4, and a second XTEN isinserted immediately downstream of amino acid 1720 corresponding to SEQID NO: 4 (full-length mature FVIII). In another example, a first XTEN isinserted immediately downstream of amino acid 403 corresponding to SEQID NO: 4, and a second XTEN is inserted immediately downstream of aminoacid 1720 corresponding to SEQ ID NO: 4. In some examples, a first XTENis inserted immediately downstream of amino acid 1656 corresponding toSEQ ID NO: 4, and a second XTEN is inserted immediately downstream ofamino acid 1720 corresponding to SEQ ID NO: 4. In other examples, afirst XTEN is inserted immediately downstream of amino acid 26corresponding to SEQ ID NO: 4, a second XTEN is inserted immediatelydownstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a thirdXTEN is inserted immediately downstream of amino acid 1720 correspondingto SEQ ID NO: 4. In yet other embodiments, a first XTEN is insertedimmediately downstream of amino acid 403 corresponding to SEQ ID NO: 4,a second XTEN is inserted immediately downstream of amino acid 1656corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediatelydownstream of amino acid 1720 corresponding to SEQ ID NO: 4. In stillother embodiments, a first XTEN is inserted between amino acids 403 and404 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediatelydownstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a thirdXTEN is inserted immediately downstream of amino acid 1720 correspondingto SEQ ID NO: 4. In certain embodiments, a first XTEN is insertedimmediately downstream of amino acid 26 corresponding to SEQ ID NO: 4(full-length mature FVIII), a second XTEN is inserted immediatelydownstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a thirdXTEN is inserted immediately downstream of amino acid 1900 correspondingto SEQ ID NO: 4. In some embodiments, a first XTEN is insertedimmediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, asecond XTEN is inserted immediately downstream of amino acid 1656corresponding to SEQ ID NO: 2, a third XTEN is inserted immediatelydownstream of amino acid 1720 corresponding to SEQ ID NO: 4, and afourth XTEN is inserted immediately downstream of amino acid 1900corresponding to SEQ ID NO: 4. In another example, an XTEN is insertedimmediately downstream of amino acid 745 corresponding to SEQ ID NO: 4.In an additional example, a first XTEN is inserted immediatelydownstream of amino acid 1656 corresponding to SEQ ID NO: 4 and a secondXTEN is inserted immediately downstream of amino acid 1900 correspondingto SEQ ID NO: 4. In some embodiments, a first XTEN is insertedimmediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, asecond XTEN is inserted immediately downstream of amino acid 1656corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediatelydownstream of amino acid 1900 corresponding to SEQ ID NO: 4. In anotherexample, a first XTEN is immediately inserted downstream of amino acid403 corresponding to SEQ ID NO: 4 and a second XTEN is insertedimmediately downstream of amino acid 745 corresponding to SEQ ID NO: 4.In some embodiments, a first XTEN is inserted immediately downstream ofamino acid 745 of corresponding to SEQ ID NO: 4, and a second XTEN isinserted immediately downstream of amino acid 1900 corresponding to SEQID NO: 4. In some embodiments, a first XTEN is inserted immediatelydownstream of amino acid 18 corresponding to SEQ ID NO: 4, and a secondXTEN is inserted immediately downstream of amino acid 745 correspondingto SEQ ID NO: 4.

In some embodiments, the FVIII protein is a dual chain FVIII isoform. Insome embodiments, the FVIII protein is a single chain FVIII isoform.

In some embodiments, the XTEN that is inserted is SEQ ID NO: 39 (AE288).In some examples, the XTENs that are inserted are SEQ ID NOs: 38 and 37(AG144 and AE144). In some examples, the XTENs that are inserted are SEQID NOs: 37, 38 and 37 (AE144, AG144, and AE144). In some embodiments.the XTENs that are inserted are SEQ ID NOs: 37 and 40 (AE144 and AE288).In some embodiments, the XTENs that are inserted are AE42 (SEQ ID NO:36), AE72 (SEQ ID NO: 127), AE144_2A (SEQ ID NO: 128), AE144_3B (SEQ IDNO: 129), AE144_4A (SEQ ID NO: 130), AE144_5A (SEQ ID NO: 131), AE144_6B(SEQ ID NO: 132), AG144_A (SEQ ID NO: 133), AG144_B (SEQ ID NO: 134),AG144_C (SEQ ID NO: 135), AG144_F (SEQ ID NO: 136), AE864 (SEQ ID NO:43), AE576 (SEQ ID NO: 41), AE288 (SEQ ID NO: 39), AE288_2 (SEQ ID NO:137), AE144 (SEQ ID NO: 37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO:42), AG288 (SEQ ID NO: 40), AG144 (SEQ ID NO: 38), and any combinationsthereof.

The FVIII protein useful in the invention can comprise B domain or aportion thereof, e.g., SQ B domain deleted FVIII. In one embodiment, theFVIII protein comprises single chain FVIII. In another embodiment, thesingle chain FVIII contains at least one amino acid substitution at aresidue corresponding to residue 1648, residue 1645, or both offull-length mature Factor VIII polypeptide (SEQ ID NO: 4) or residue754, residue 751, or both of SQ BDD Factor VIII (SEQ ID NO: 6). In otherembodiments, the amino acid substitution is an amino acid other thanarginine. In some embodiments, the FVIII protein comprises a heavy chainof FVIII and a light chain of FVIII, wherein the heavy chain and thelight chain are associated with each other by a metal bond.

The FVIII protein can have a low affinity to or does not bind to alow-density lipoprotein receptor-related protein (LRP), e.g., bycontaining at least one amino acid substitution that lowers the affinityto or eliminates the binding to the LRP. Such at least one amino acidsubstitution can be at a residue corresponding to residue 471, residue484, residue 487, residue 490, residue 497, residue 2092, residue 2093or two or more combinations thereof of full-length mature FVIII. In aparticular embodiment, the amino acid substitution at residue 471, 484,or 497 is an amino acid other than arginine, the amino acid substitutionat residue 487 is an amino acid other than tyrosine, the amino acidsubstitution at residue 2092 is an amino acid other than lysine, or theamino acid substitution at residue 2093 is an amino acid other thanphenylalanine.

In some embodiments, the FVIII protein contains at least one amino acidsubstitution, which induces the FVIII protein to be more stable than aFVIII protein without the substitution. Such substitutions can belocated in the A2 domain and the A3 domain of the FVIII protein, e.g.,at a residue corresponding to residue 664, residue 1826, residue 662,residue 1828, or two or more combinations thereof of full-length matureFVIII.

The VWF fragment useful for the present invention comprises a D′ domainand D3 domain, which together are capable of binding to FVIII. The VWFfragment can comprise the amino acid sequence of the D′ domain is atleast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764to 866 of SEQ ID NO: 2 and/or the amino acid sequence of the D3 domainis at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to aminoacids 867 to 1240 of SEQ ID NO: 2. In one embodiment, the VWF fragmentis a monomer. In another embodiment, the VWF fragment comprises at leasttwo VWF fragments, at least three VWF fragments, at least four VWFfragments, at least five VWF fragments, or at least six VWF fragments.In one embodiment, the two or more VWF fragments may be identical orthey may be different. The VWF fragment can comprise an amino acid atleast 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764to 1240 of SEQ ID NO: 2. The VWF fragment may consist essentially of orconsist of amino acids 764 to 1240 of SEQ ID NO: 2. In certainembodiments, the VWF fragment can contain at least one amino acidsubstitution at a residue corresponding to residue 1099, residue 1142,or both residues 1099 and 1142 of SEQ ID NO: 2. In other embodiments,the VWF fragment further comprises the D1 domain, the D2 domain, or theD1 and D2 domains of VWF.

The VWF fragment may further comprise a VWF domain selected from thegroup consisting of the A1 domain, the A2 domain, the A3 domain, the D4domain, the B1 domain, the B2 domain, the B3 domain, the C1 domain, theC2 domain, the CK domain, one or more fragments thereof, and anycombinations thereof. For example, the VWF fragment can consistessentially of or consist of: (1) the D′ and D3 domains of VWF orfragments thereof; (2) the D1, D′, and D3 domains of VWF or fragmentsthereof; (3) the D2, D′, and D3 domains of VWF or fragments thereof, (4)the D1, D2, D′, and D3 domains of VWF or fragments thereof; or (5) theD1, D2, D′, D3, and A1 domains of VWF or fragments thereof. In someembodiments, the VWF fragment further comprises a signal peptide of VWFor FVIII which is operably linked to the VWF fragment.

One or more of the linkers useful in the invention have a length of atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400,1600, 1800, or 2000 amino acids. In some embodiments, one or more of thelinkers have a length of about 1 to about 2000 amino acids. In oneembodiment, one or more of the linkers have a length of at least about20, 35, 42, 48, 73, 75, 95, 98, 144, 288, 324, 333, 576, or 864 aminoacids. In another embodiment, one or more of the linkers comprise agly/ser peptide, an XTEN sequence, or both. Examples of the gly/serpeptide include, but are not limited to, a formula of (Gly₄Ser)_(n) (SEQID NO: 139) or S(Gly₄Ser)_(n) (SEQ ID NO: 140), wherein n is a positiveinteger selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9and 10. For example, the (Gly₄Ser)_(n) linker can be (Gly₄Ser)₃ (SEQ IDNO: 63) or (Gly₄Ser)₄ (SEQ ID NO: 138). In one embodiment, the linkercomprises at least one first cleavage site at the N-terminus of thelinker, at least one second cleavage site at the C-terminus of thelinker, or both. In another embodiment, the linker comprises 20 aminoacids, 35 amino acids, 48 amino acids, 73 amino acids, or 95 amino acidsthrombin cleavable linker. The cleavable linkers can comprise one ormore of the cleavage sites by a protease selected from the groupconsisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factorIXa, factor Xa, factor IIa (thrombin), Elastase-2, Granzyme-B, TEV,Enterokinase, Protease 3C, Sortase A, MMP-12, MMP-13, MMP-17, andMMP-20, e.g., TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 8). Non-limitingexamples of one or more of the cleavage sites comprise an amino acidsequence selected from the group consisting of RRRR (SEQ ID NO: 9),RKRRKR (SEQ ID NO: 10), RRRRS (SEQ ID NO: 11), TQSFNDFTR (SEQ ID NO:12), SVSQTSKLTR (SEQ ID NO: 13), DFLAEGGGVR (SEQ ID NO: 14), TTKIKPR(SEQ ID NO: 15), LVPRG (SEQ ID NO: 16), ALRPR (SEQ ID NO: 17), KLTRAET(SEQ ID NO: 18), DFTRVVG (SEQ ID NO: 19), TMTRIVGG (SEQ ID NO: 20),SPFRSTGG (SEQ ID NO: 21), LQVRIVGG (SEQ ID NO: 22), PLGRIVGG (SEQ IDNO:23), IEGRTVGG (SEQ ID NO: 24), LTPRSLLV (SEQ ID NO: 25), LGPVSGVP(SEQ ID NO: 26), VAGDSLEE (SEQ ID NO: 27), GPAGLGGA (SEQ ID NO: 28),GPAGLRGA (SEQ ID NO: 29), APLGLRLR (SEQ ID NO: 30), PALPLVAQ (SEQ ID NO:31), ENLYFQG (SEQ ID NO: 32), DDDKIVGG (SEQ ID NO: 33), LEVLFQGP (SEQ IDNO: 34), and LPKTGSES (SEQ ID NO: 35). In some embodiments, the firstcleavage site and the second cleavage site are identical or different.

The XTEN sequence useful for the invention can be selected from thegroup consisting of AE42 (SEQ ID NO: 36), AE144 (SEQ ID NO: 37), AG144(SEQ ID NO: 38), AE288 (SEQ ID NO: 39), AG288 (SEQ ID NO: 40), AE576(SEQ ID NO: 41). AG576 (SEQ ID NO: 42), AE864 (SEQ ID NO: 43), AE72 (SEQID NO: 127), AE144_2A (SEQ ID NO: 128), AE144_3B (SEQ ID NO: 129),AE144_4A (SEQ ID NO: 130), AE144_5A (SEQ ID NO: 131), AE144_6B (SEQ IDNO: 132), AG144_A (SEQ ID NO: 133), AG144_B (SEQ ID NO: 134), AG144_C(SEQ ID NO:135), AG144_F (SEQ ID NO: 136), AE288_2 (SEQ ID NO: 137), orAG864 (SEQ ID NO: 44). In a particular embodiment, the XTEN sequencecomprises AE288 or AG288.

The chimeric protein of the invention can be polysialylated, pegylated,or hesylated.

The present invention is also directed to a polynucleotide or a set ofpolynucleotides encoding the chimeric protein. The polynucleotide canfurther comprise a polynucleotide chain, which encodes PC5 or PC7. Theinvention is also directed to a vector comprising the polynucleotide orthe set of polynucleotides and one or more promoter operably linked tothe polynucleotide or the set of polynucleotides. The vector can furthercomprise an additional vector, which comprises a polynucleotide chainencoding PC5 or PC7. The invention is also drawn to a host cellcomprising the polynucleotide or the vector. The host cell can be amammalian cell, e.g., HEK293 cell, CHO cell, or BHK cell. In someembodiments, the PC5 or PC7 of the host cell cleaves the D1D2 domains ofVWF.

The invention is also directed to a pharmaceutical compositioncomprising the chimeric protein, the polynucleotide, the vector, or thehost cell, and a pharmaceutically acceptable carrier. The composition ofthe invention thus has an extended half-life compared to wild type FVIIIprotein. The half-life of the FVIII protein is extended at least about1.5 times, at least about 2 times, at least about 2.5 times, at leastabout 3 times, at least about 4 times, at least about 5 times, at leastabout 6 times, at least about 7 times, at least about 8 times, at leastabout 9 times, at least about 10 times, at least about 11 times, or atleast about 12 times longer than wild type FVIII. The half-life ofFactor VIII is at least about 17 hours, at least about 18 hours, atleast about 19 hours, at least about 20 hours, at least about 21 hours,at least about 22 hours, at least about 23 hours, at least about 24hours, at least about 25 hours, at least about 26 hours, at least about27 hours, at least about 28 hours, at least about 29 hours, at leastabout 30 hours, at least about 31 hours, at least about 32 hours, atleast about 33 hours, at least about 34 hours, at least about 35 hours,at least about 36 hours, at least about 48 hours, at least about 60hours, at least about 72 hours, at least about 84 hours, at least about96 hours, or at least about 108 hours.

The composition of the present invention can be administered by a routeselected from the group consisting of topical administration,intraocular administration, parenteral administration, intrathecaladministration, subdural administration and oral administration. In oneembodiment, the composition is administered via parenteraladministration, e.g., intravenous or subcutaneous administration. Thecomposition of the invention is useful to treat a bleeding disease orcondition in a subject in need thereof. The bleeding disease orcondition is selected from the group consisting of a bleedingcoagulation disorder, hemarthrosis, muscle bleed, oral bleed,hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, traumacapitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, bleeding in the illiopsoas sheathand any combinations thereof. In one embodiment, the subject treatedwith the chimeric protein is scheduled to undergo a surgery. In anotherembodiment, the treatment is prophylactic or on-demand.

The invention is also directed to a method of preventing or inhibitingbinding of a FVIII protein with endogenous VWF comprising adding aneffective amount of the chimeric protein, the polynucleotide vector, thehost cell, or the composition to a subject in need thereof, wherein theVWF fragment binds to the FVIII protein and thus prevents or inhibitsbinding of endogenous VWF. The present invention is further directed toa method of extending or increasing the half-life of the FVIII protein,wherein the method comprises administering an effective amount of thechimeric protein, the polynucleotide, the vector, the host cell, or thecomposition to a subject in need thereof, wherein the VWF fragment bindsto the FVIII protein and thus extends or increases the half-life of theFVIII protein. Also provided is a method of preventing or inhibitingclearance of a FVIII protein from a cell, wherein the method comprisesadministering an effective amount of the chimeric protein, thepolynucleotide, the vector, the host cell, or the composition to a cellcomprising a FVIII protein or a polynucleotide encoding the FVIIIprotein, wherein the protein having VWF activity binds to the FVIIIprotein. The subject useful for the present methods is an animal, e.g.,a human, e.g., a patient suffering from hemophilia A.

The present invention also provides a method of treating a bleedingdisease or disorder in a subject in need thereof comprisingadministering an effective amount of the chimeric protein, thepolynucleotide, the vector, the host cell, or the composition, whereinthe bleeding disease or disorder is selected from the group consistingof a bleeding coagulation disorder, hemarthrosis, muscle bleed, oralbleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma,trauma capitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, and bleeding in the illiopsoassheath. The treatment can be prophylactic or on-demand. In oneembodiment, the effective amount is 0.1 μg/kg to 500 mg/kg.

The invention also includes a method of making a chimeric protein,comprising transfecting one or more host cell with the polynucleotide orthe vector and expressing the chimeric protein in the host cell.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A-D. Schematic diagrams of VWF fragments. FIG. 1A shows threeexemplary VWF fragments useful for the invention, e.g., VWF-002,VWF-010, and VWF-013. VWF-002 contains amino acids 1 to 477 of SEQ IDNO: 124 (amino acids 764 to 1240 of SEQ ID NO: 2) and is synthesizedwithout the pre/propeptide sequences. VWF-010 contains the D1D2 domainsin addition to the D′D3 domains. VWF-013 contains the D1D2D′D3 domainsin addition to alanine residues substituting cysteines at residues 336and 379 of SEQ ID NO: 123 FIG. 1B shows VWF-031, which contains theD1D2D′D3 domains fused to an Ig constant region or a portion thereof,e.g., an Fc region, by a cleavable linker, e.g., a 48 amino acidsthrombin cleavable linker. FIG. 1C shows VWF-025, which is a nucleotidesequence encoding D1D2D′D3 domains contained in pLIVE vector, andVWF-029, which is a nucleotide sequence encoding D1D2D′D3 domains withtwo amino acid substitutions, C336A and C379A, in pLIVE vector. FIG. 1Dshows full-length VWF fragment comprising propeptide (the D1 and D2domains) and mature subunits (the D′, D3, A1, A2, A3, D4, B1-3, C1-2domains). The VWF fragment is about 250 kDa protein and forms multimers(>20 MDa) by disulfide bonding. The VWF fragment associates with FVIII(95-98%) in non-covalent complex and then extends half-life of FVIII byprotecting FVIII from protease cleavage/activation, stabilizing heavy &light chain, and preventing clearance of FVIII by scavenger receptors.The VWF fragment also can limit half-life of FVIII by clearance ofFVIII-VWF complex through VWF receptors and preventing pinocytosis andrecycling of rFVIIIFc.

FIG. 2. Pharmacokinetic profile of rFVIII-XTEN (rFVIII-AE288 orrFVIII-288AE) in VWF D′D3 expression mice or in FVIII and VWF doubleknockout (DKO) mice. FIG. 2A shows the timeline of hydrodynamicinjection (HDI) of the D′D3 domain encoding plasmid DNA (VWF-025) (day−5), intravenous dosing of rFVIII-XTEN AE288 (day 0), and PK samplecollection (day 5). FIG. 2B shows FVIII activity measured by a FVIIIchromogenic assay after IV dosing of rFVIII-XTEN288 in D1D2D′D3 mice(inverted triangle) and rFVIII-XTEN288 in DKO mice (diamond). FIG. 2Cshows the D′D3 plasma level (ng/mL) after administration of VWF-025. TheX axis represents time in hours.

FIG. 3. Schematic diagram of exemplary VWF:FVIII heterodimer constructs.The constructs have the common structure represented as formulaFVIII-F1-L1-V-X-L2-F2, but contain examples of different variablelinkers. The construct (FVIII-161) shown contains a heterodimeric FVIII(the heavy chain and the light chain are associated by a metal bond)linked to a first Fc region and a VWF fragment, which is the D′ and D3domains of VWF (i.e., amino acids 1 to 477 of SEQ ID NO: 2 with aminoacid substitutions C336A and C379A) linked to an XTEN sequence, which isfurther linked to a cleavable linker and a second Fc region. The XTENsequence contained in FVIII-161 is an XTEN AE288 sequence, and thelinker is a thrombin cleavable linker, which has 35 amino acids. InFVIII-161, the FVIII protein linked to the first Fc region is linked tothe VWF fragment by a processable linker. Upon expression, theprocessable linker can be cleaved by an intracellular processing enzyme,thus making the construct three polypeptide chains associated with eachother.

FIG. 4 is schematic diagrams of FVIII-VWF heterodimer or monomerexamples. FVIII-168, FVIII-175, FVIII-172, FVIII-174, and FVIII170.Construct FVIII-168 comprises a single chain FVIII sequence (having analanine residue substitute the arginine residues at residues 1645 and1648) linked to a first Fc region, which is then fused to a VWF fragmentlinked to a second Fc region by a thrombin cleavable linker, which has48 amino acids. AE288 XTEN is inserted in the B domain of the singlechain FVIII sequence. The linkage between the first Fc region and theVWF fragment comprises a linker that is capable of being cleaved by anintracellular processing enzyme, i.e., processable linker. ConstructFVIII-175 comprises a single chain FVIII (having an alanine residuesubstitute the arginine residues at residues 1645 and 1648) linked toAE288 XTEN and a first Fc region, which is linked to a second Fc regionby a linker, e.g., a processable linker. AE288 XTEN is inserted in the Bdomain of the single chain FVIII sequence. Construct FVIII-172 comprisestwo polypeptide chains, a first chain comprising a heavy chain FVIIIsequence fused to AE288 XTEN, a second chain comprising a light chainFVIII sequence, a first Fc region, a linker (e.g., a processablelinker), a VWF fragment, a thrombin cleavable linker (e.g., 48 aminoacids), and a second Fc region. Construct FVIII-174 comprises twopolypeptide chains, a first chain comprising a heavy chain FVIIIsequence fused to AE288 XTEN and a second chain comprises a light chainFVIII, a first Fc region, a linker (e.g., a processable linker), and asecond Fc region. Construct FVIII-170 comprises a VWF fragment, AE288XTEN, a linker (e.g., a thrombin cleavable linker, which is 35 aminoacids in length), and a single chain FVIII sequence.

FIG. 5. Pharmacokinetic profile of FVIII/VWF heterodimers containing anXTEN sequence in combination with an Fc region. Constructs FVIII-161,FVIII-168, and FVIII-172 were administered to FVIII:VWF double knockout(DKO) mice by Hydrodynamic injection (HDI) at 100 ug/mouse dose.Construct FVIII-170 was administered to FVIII:VWF DKO mice by HDI at 50μg/mouse dose. The post-HDI plasma FVIII activity was analyzed by FVIIIchromogenic assay for 24 hr post-HDI. The FVIII activity of theFVIII:VWF heterodimers containing an XTEN sequence and Fc domains wascompared with the FVIII activity of BDD-FVIII without the VWF fragment,XTEN sequence, and Fc domains.

FIG. 6. Schematic diagrams of FVIII-VWF heterodimer examplesco-transfection system. FIG. 6A. Construct FVIII-169 contains thefull-length FVIII sequence (with an alanine residue substituting thearginine residues at 1645 and 1648 and with an XTEN sequence inserted inthe single chain FVIII sequence), which is linked to an Fc region.VWF-031 contains the D1D2D′D3 fragment (with an alanine residuesubstituting the cysteine residues at 336 and 379) which is linked toanother Fc region with a 48 thrombin cleavable linker. Afterintracellular processing, construct FVIII-169 produces a full lengthsingle chain FVIII (SCFVIII) fused to one Fc fragment and an XTENsequence, and construct VWF-031 produces a 477 amino acid D′D3 fragmentlinked to another Fc fragment. Two covalent bonds can be formed betweenthe Fc fragments that are linked to the SC FVIII or the D′D3 fragment,this in turn allows a non-covalent association of FVIII and D′D3. FIG.6B. Construct FVIII-173 contains a heterodimeric FVIII sequence, a heavychain FVIII sequence linked to an XTEN sequence and a light chain FVIIIsequence linked to an Fc region. VWF-031 is described above. Afterintracellular processing, construct FVIII-173 produces a heterodimericprotein, a heavy chain FVIII fused to an XTEN sequence, a light chainFVIII fused to one Fc fragment, and construct VWF-031 produces a 477amino acid D′D3 fragment linked to another Fc fragment. Two covalentbonds can be formed between the Fc fragments that are linked to thelight chain FVIII or the D′D3 fragment, this in turn allows anon-covalent association of FVIII and D′D3.

FIG. 7. Binding Affinity of Exemplary FVIII:VWF containing an XTENsequence and Fc domains to immobilized hVWF in Octet assay. The bindingaffinity for FVIII-169/VWF-031 and FVIII-057 (rFVIIIFc) fused toimmobilized hVWF was tested using biolayer interferometry basedmeasurements (Octet assay). FIG. 7A shows binding response in nanomolesof FVIII169 and FVIIIFc drug substance (a positive control) toimmobilized hVWF. FIG. 7B shows binding response of human IgG1 (anegative control) to immobilized human VWF.

FIG. 8. Pharmacokinetic (PK) profile of FVIII-169 in HemA and FVIII:VWFdouble knockout (DKO) mice. FIG. 8A shows the PK profile ofFVIII-169/VWF-031 and FVIIIFc in HemA mice. HemA mice were treated witha single intravenous dose of FVIII-169/VWF-031 at 200 IU/kg. Plasmasamples collected from the mice were tested by FVIII chromogenic assay.Half-life of FVIII-169/VWF-031 was calculated using WinNonlin program.FIG. 8B shows the PK profile of FVIII-169/VWF-031, FVIII-169/Fc, andFVIIIFc in FVIII/VWF DKO mice.

FIG. 9. PK profile of FVIII-XTEN variants in D′D3 expressing FVIII/VWFDKO mice. FIG. 9A shows comparison of the PK profile of the FVIII-XTENvariants, FVIII with one XTEN, FVIII with two XTENs, and FVIII withthree XTENs. One, two, or three XTENs were inserted in various portionsof FVIII including C-terminus and B-domain. CT indicates that an XTEN islinked to the C-terminus of FVIII. Insertion site B/CT indicates thatone XTEN is inserted between amino acid residue 745 and amino acidresidue 746 of the FVIII protein and another XTEN is linked to theC-terminus of the FVIII protein. The amino acid residue numberingcorresponds to the SQ BDD FVIII protein sequence. Insertion site1900/B/CT indicates that a first XTEN is inserted between amino acidresidue 1900 and amino acid residue 1901 of FVIII, a second XTEN isinserted between amino acid residue 745 and amino acid residue 746 ofFVIII, and a third XTEN is linked to the C-terminus of FVIII. The mousestrain used to administer the FVIII-XTEN variants is a DKO mouse strainexpressing D′D3 domains. FIG. 9B shows the PK profile of FVIII-XTEN withthree XTEN insertions. The FVIII-XTEN (1900/B/CT) variant wasadministered to either the FVIII/VWF DKO mice or HemA mice. Thehalf-life of FVIII-XTEN (1900/B/CT) is compared.

FIG. 10. FVIII activity of FVIIIFc (hollow triangle), FVIII169:Fc(filled circle), and FVIII169:VWF31 (hollow triangle) in mouse DKOplasma measured by chromogenic assay. FVIII:Fc contains a dual-chainFVIII (Heavy chain and Light chain) fused to an Fc dimer (i.e.,monomer-dimer hybrid). FVIII169 is described above (containing AE288 inthe B domain, immediately downstream of amino acid 745 corresponding tomature FVIII sequence). FVIII169:Fc contains FVIII169 fused to an Fcdimer. FVIII169:VWF31 contains VWF31 in addition to the Fc dimer,FVIII169 fused to the first Fc region and VWF31 fused to the second Fcregion, wherein the first Fc region and the second Fc region form acovalent bond, e.g., one or more disulfide bonds.

FIG. 11. Effects of Fc, XTEN, and VWF-D′D3 fragments on FVIII half-lifeextension. BDD-FVIII (REFACTO®) (square), FVIIFc (circle), FVIII169/Fc(triangle), and FVIII169NWF031 (inverted triangle) were administered toFVIII and VWF double knockout (DKO) mice. The FVIII activity wasmeasured by chromogenic assay, and the half-life was calculated usingthe WinNonlin-Phoenix program. X-axis shows time, and the Y-axis showsthe FVIII plasma activity in mU/mL.

FIG. 12A-C. Effects of different XTENs in rFVIII-XTEN/VWF heterodimer inHemA mice. FIG. 12A shows the FVIII plasma activity normalized to 5 minvalue (%) of two XTENs inserted immediately downstream of residues 1900and 1656 corresponding to mature FVIII sequence (i.e., FVIII-195 (dualchain FVIII isoform) and FVIII-199 (single chain FVIII isoform)),compared to FVIII-169 containing an XTEN immediately downstream ofresidue 745 corresponding to mature FVIII sequence. FVIII-169NWF-031(filled circle), FVIII-199/VWF-031 (filled square), and FVIII-195NWF031(hollow square) were administered in HemA mice to measure the FVIIIplasma activity. FIG. 12B shows the half-life extension effect of thesecond XTEN insertion immediately downstream of residues 403 (A2 domain)and 745 (B domain) (i.e., FVIII-203) and residues 745 (B domain) and1900 (A3 domain) (FVIII-204) corresponding to mature FVIII sequencecompared to FVIII-169 (an XTEN insertion in B domain only).FVIII-204NWF031 (filled triangle), FVIII-169NWF-031 (filled circle),FVIII-203NWF-031 (filled square), and scBDD-FVIII (hollow diamond) wereadministered to HemA mice. The X-axis shows FVIII plasma activitynormalized to 5 min value (%), and the y-axis shows time in hours. FIG.12C shows the half-life extension effect of the two XTEN insertionsimmediately downstream of residues 18 (A1 domain) and 745 (B domain)(i.e., FVIII-205) compared to FVIII-169 (a single XTEN insertion in theB domain) and single chain FVIII without any Fc regions or any XTENs(i.e., FVIII-207). FIG. 12C additionally shows the half-life extensioneffect of three XTEN insertions incorporated immediately downstream ofresidues 26 (A1 domain), 1656 (A3 domain), and 1900 (A3 domain) (i.e.,FVIII-201) compared to FVIII-169 (a single XTEN insertion immediatelydownstream of residue 745). FVIII-205NWF-031 (filled square),FVIII-201NWF-031 (inverted triangle), FVIII-169NWF-031 (filled circle),and FVIII-207 (hollow diamond) were administered to HemA mice. The FVIIIplasma activity normalized to 5 min value (%) (X-axis) was measured overtime in hours (Y-axis).

FIG. 13. FVIII activity of rFVIII-XTEN/VWF-XTEN heterodimer in FVIII/VWFDKO mice. FVIII activity of plasma samples was analyzed by FVIIIchromogenic assay, and the regression curve of plasma FVIII activity(X-axis) as a function of time (Y-axis) was plotted. FVIII-155(scFVIIIFc without any XTENs) was co-expressed with VWF-034 (VWF-Fc withAE 288 XTEN plus a 35 residue thrombin cleavable linker). The half-lifeof FVIII-155/VWF-034 was compared with that of FVIII-169/VWF-031, whichhas a AE 288 XTEN inserted into the B domain junction (immediatelydownstream of residue 745 corresponding to mature FVIII polypeptide) ofFVIII.

FIG. 14A-H. Schematic diagrams of various rFVIII-XTEN/VWF constructs.These constructs are also described in other sections herein. FIG. 14Ashows single chain B domain deleted FVIII protein (sometimes indicatedherein as scBDD-FVIII). The scBDD-FVIII constructs contain twosubstitutions at residues 1645 and 1648 from Arg to Ala. FIG. 14B showstwo polypeptide chain construct (FVIII155NWF031), the first chaincomprising single chain FVIII linked to an Fc region without any XTENSand the second chain comprising the VWF D′D3 fragment linked to an Fcregion. This construct is used as a control. FIG. 14C shows twopolypeptide chain construct (FVIII199NWF031), the first chain comprisingsingle chain FVIII linked to an Fc region, in which a first XTEN isinserted immediately downstream of residue 1900 corresponding to matureFVIII sequence and a second XTEN is inserted immediately downstream ofresidue 1656 corresponding to mature FVIII sequence, and the secondchain comprising the VWF D′D3 fragment linked to an Fc region. FIG. 14Dshows two polypeptide chain construct (FVIII201NWF031), the first chaincomprising single chain FVIII protein linked to an Fc region, in which afirst XTEN is inserted immediately downstream of residue 26corresponding to mature FVIII sequence, a second XTEN is insertedimmediately downstream of residue 1656 corresponding to mature FVIIIsequence, and a third XTEN is inserted immediately downstream of residue1900 corresponding to mature FVIII sequence, and the second chaincomprising the VWF D′D3 fragment linked to an Fc region. FIG. 14E showstwo polypeptide chain constructs (FVIII169/VWF031), the first chaincomprising single chain FVIII protein linked to an Fc region, in whichan XTEN is inserted immediately downstream of residue 745 (indicated as“B”) corresponding to mature FVIII sequence, and the second chaincomprising the VWF D′D3 fragment linked to an Fc region. FIG. 14F showstwo polypeptide chain construct (FVIII203NWF031), the first chaincomprising single chain FVIII protein, in which a first XTEN is insertedat residue 745 (“B”) corresponding to mature FVIII sequence and a secondXTEN is inserted at residue 1900 corresponding to mature FVIII sequence,and the second chain comprising the VWF D′D3 fragment linked to an Fcregion. FIG. 14G shows two polypeptide chain construct (FVIII204NWF031),the first chain comprising single chain FVIII protein linked to an Fcregion, in which a first XTEN is inserted immediately downstream ofresidue 403 corresponding to mature FVIII sequence and a second XTEN isinserted immediately downstream of residue 745 (“B”) corresponding tomature FVIII sequence, and a second chain comprising the VWF D′D3fragment linked to an Fc region. FIG. 14H shows two polypeptide chainconstruct (FVIII205NWF031), the first chain comprising single chainFVIII, in which a first XTEN is inserted immediately downstream ofresidue 18 corresponding to mature FVIII sequence and a second XTEN isinserted immediately downstream of residue 745 (“B”) corresponding tomature FVIII sequence, and the second chain comprising the VWF D′D3fragment linked to an Fc region.

FIG. 15. FVIII activity of rFVIII-XTEN/VWF and BDD-FVIII in FVIII/VWFDKO mice. FVIII activity of plasma samples was analyzed by FVIIIchromogenic assay, and the regression curve of plasma FVIII activity(X-axis) as a function of time (Y-axis) was plotted. The half-life ofrFVIII-XTEN/VWF (FVIII-205/VWF-031) was compared with that of BDD-FVIIIand rFVIIIFc.

FIG. 16. Efficacy of FVIII-XTEN-Fc:VWF-Fc heterodimers in HemA miceusing tail clip bleeding model. The HemA mice tail clip bleeding modelwas used to compare the efficacy of FVIII169NWF034, FVIII205NWF031, andBDD-FVIII. The median blood loss in ml for 200 IU/kg of FVIII169NWF034and FVIII205/VWF031 is compared with 200 IU/kg of BDD-FVIII, 65 IU/kg ofBDD-FVIII, 20 IU/kg of BDD-FVIII, and vehicle.

DETAILED DESCRIPTION OF THE INVENTION Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a nucleotide sequence,” is understood torepresent one or more nucleotide sequences. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

The term “polynucleotide” or “nucleotide” is intended to encompass asingular nucleic acid as well as plural nucleic acids, and refers to anisolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA)or plasmid DNA (pDNA). In certain embodiments, a polynucleotidecomprises a conventional phosphodiester bond or a non-conventional bond(e.g., an amide bond, such as found in peptide nucleic acids (PNA)). Theterm “nucleic acid” refers to any one or more nucleic acid segments,e.g., DNA or RNA fragments, present in a polynucleotide. By “isolated”nucleic acid or polynucleotide is intended a nucleic acid molecule, DNAor RNA, which has been removed from its native environment. For example,a recombinant polynucleotide encoding a Factor VIII polypeptidecontained in a vector is considered isolated for the purposes of thepresent invention. Further examples of an isolated polynucleotideinclude recombinant polynucleotides maintained in heterologous hostcells or purified (partially or substantially) from otherpolynucleotides in a solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, a polynucleotide or a nucleic acid can include regulatoryelements such as promoters, enhancers, ribosome binding sites, ortranscription termination signals.

As used herein, a “coding region” or “coding sequence” is a portion ofpolynucleotide which consists of codons translatable into amino acids.Although a “stop codon” (TAG, TGA, or TAA) is typically not translatedinto an amino acid, it may be considered to be part of a coding region,but any flanking sequences, for example promoters, ribosome bindingsites, transcriptional terminators, introns, and the like, are not partof a coding region. The boundaries of a coding region are typicallydetermined by a start codon at the 5′ terminus, encoding the aminoterminus of the resultant polypeptide, and a translation stop codon atthe 3′ terminus, encoding the carboxyl terminus of the resultingpolypeptide. Two or more coding regions of the present invention can bepresent in a single polynucleotide construct, e.g., on a single vector,or in separate polynucleotide constructs, e.g., on separate (different)vectors. It follows, then, that a single vector can contain just asingle coding region, or comprise two or more coding regions, e.g., asingle vector can separately encode a binding domain-A and a bindingdomain-B as described below. In addition, a vector, polynucleotide, ornucleic acid of the invention can encode heterologous coding regions,either fused or unfused to a nucleic acid encoding a binding domain ofthe invention. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain.

Certain proteins secreted by mammalian cells are associated with asecretory signal peptide which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that signal peptides are generally fused to the N-terminus of thepolypeptide, and are cleaved from the complete or “full-length”polypeptide to produce a secreted or “mature” form of the polypeptide.In certain embodiments, a native signal peptide or a functionalderivative of that sequence that retains the ability to direct thesecretion of the polypeptide that is operably associated with it.Alternatively, a heterologous mammalian signal peptide, e.g., a humantissue plasminogen activator (TPA) or mouse B-glucuronidase signalpeptide, or a functional derivative thereof, can be used.

The term “downstream” refers to a nucleotide sequence that is located 3′to a reference nucleotide sequence. In certain embodiments, downstreamnucleotide sequences relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′to a reference nucleotide sequence. In certain embodiments, upstreamnucleotide sequences relate to sequences that are located on the 5′ sideof a coding region or starting point of transcription. For example, mostpromoters are located upstream of the start site of transcription.

As used herein, the term “regulatory region” refers to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding region, and whichinfluence the transcription, RNA processing, stability, or translationof the associated coding region. Regulatory regions may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites andstem-loop structures. If a coding region is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

A polynucleotide which encodes a gene product, e.g., a polypeptide, caninclude a promoter and/or other transcription or translation controlelements operably associated with one or more coding regions. In anoperable association a coding region for a gene product, e.g., apolypeptide, is associated with one or more regulatory regions in such away as to place expression of the gene product under the influence orcontrol of the regulatory region(s). For example, a coding region and apromoter are “operably associated” if induction of promoter functionresults in the transcription of mRNA encoding the gene product encodedby the coding region, and if the nature of the linkage between thepromoter and the coding region does not interfere with the ability ofthe promoter to direct the expression of the gene product or interferewith the ability of the DNA template to be transcribed. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can also be operably associated with a coding region to direct geneproduct expression.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit B-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

The term “expression” as used herein refers to a process by which apolynucleotide produces a gene product, for example, an RNA or apolypeptide. It includes without limitation transcription of thepolynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), smallhairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNAproduct, and the translation of an mRNA into a polypeptide. Expressionproduces a “gene product.” As used herein, a gene product can be eithera nucleic acid, e.g., a messenger RNA produced by transcription of agene, or a polypeptide which is translated from a transcript. Geneproducts described herein further include nucleic acids with posttranscriptional modifications, e.g., polyadenylation or splicing, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, or proteolytic cleavage.

A “vector” refers to any vehicle for the cloning of and/or transfer of anucleic acid into a host cell. A vector may be a replicon to whichanother nucleic acid segment may be attached so as to bring about thereplication of the attached segment. A “replicon” refers to any geneticelement (e.g., plasmid, phage, cosmid, chromosome, virus) that functionsas an autonomous unit of replication in vivo, i.e., capable ofreplication under its own control. The term “vector” includes both viraland nonviral vehicles for introducing the nucleic acid into a cell invitro, ex vivo or in vivo. A large number of vectors are known and usedin the art including, for example, plasmids, modified eukaryoticviruses, or modified bacterial viruses. Insertion of a polynucleotideinto a suitable vector can be accomplished by ligating the appropriatepolynucleotide fragments into a chosen vector that has complementarycohesive termini.

Vectors may be engineered to encode selectable markers or reporters thatprovide for the selection or identification of cells that haveincorporated the vector. Expression of selectable markers or reportersallows identification and/or selection of host cells that incorporateand express other coding regions contained on the vector. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like. Examples of reporters knownand used in the art include: luciferase (Luc), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ),-glucuronidase (Gus), and the like. Selectable markers may also beconsidered to be reporters.

The term “plasmid” refers to an extra-chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

Eukaryotic viral vectors that can be used include, but are not limitedto, adenovirus vectors, retrovirus vectors, adeno-associated virusvectors, and poxvirus, e.g., vaccinia virus vectors, baculovirusvectors, or herpesvirus vectors. Non-viral vectors include plasmids,liposomes, electrically charged lipids (cytofectins), DNA-proteincomplexes, and biopolymers.

A “cloning vector” refers to a “replicon,” which is a unit length of anucleic acid that replicates sequentially and which comprises an originof replication, such as a plasmid, phage or cosmid, to which anothernucleic acid segment may be attached so as to bring about thereplication of the attached segment. Certain cloning vectors are capableof replication in one cell type, e.g., bacteria and expression inanother, e.g., eukaryotic cells. Cloning vectors typically comprise oneor more sequences that can be used for selection of cells comprising thevector and/or one or more multiple cloning sites for insertion ofnucleic acid sequences of interest.

The term “expression vector” refers to a vehicle designed to enable theexpression of an inserted nucleic acid sequence following insertion intoa host cell. The inserted nucleic acid sequence is placed in operableassociation with regulatory regions as described above.

Vectors are introduced into host cells by methods well known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a DNA vector transporter.

“Culture,” “to culture” and “culturing,” as used herein, means toincubate cells under in vitro conditions that allow for cell growth ordivision or to maintain cells in a living state. “Cultured cells,” asused herein, means cells that are propagated in vitro.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” canbe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide can be derived from a natural biological source or producedrecombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It can be generated in any manner,including by chemical synthesis.

An “isolated” polypeptide or a fragment, variant, or derivative thereofrefers to a polypeptide that is not in its natural milieu. No particularlevel of purification is required. For example, an isolated polypeptidecan simply be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for the purpose of the invention, as are nativeor recombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included in the present invention are fragments or variants ofpolypeptides, and any combination thereof. The term “fragment” or“variant” when referring to polypeptide binding domains or bindingmolecules of the present invention include any polypeptides which retainat least some of the properties (e.g., FcRn binding affinity for an FcRnbinding domain or Fc variant, coagulation activity for an FVIII variant,or FVIII binding activity for the VWF fragment) of the referencepolypeptide. Fragments of polypeptides include proteolytic fragments, aswell as deletion fragments, in addition to specific antibody fragmentsdiscussed elsewhere herein, but do not include the naturally occurringfull-length polypeptide (or mature polypeptide). Variants of polypeptidebinding domains or binding molecules of the present invention includefragments as described above, and also polypeptides with altered aminoacid sequences due to amino acid substitutions, deletions, orinsertions. Variants can be naturally or non-naturally occurring.Non-naturally occurring variants can be produced using art-knownmutagenesis techniques. Variant polypeptides can comprise conservativeor non-conservative amino acid substitutions, deletions or additions.

The term “VWF fragment” or “VWF fragments” used herein means any VWFfragments that interact with FVIII and retain at least one or moreproperties that are normally provided to FVIII by full-length VWF, e.g.,preventing premature activation to FVIIIa, preventing prematureproteolysis, preventing association with phospholipid membranes thatcould lead to premature clearance, preventing binding to FVIII clearancereceptors that can bind naked FVIII but not VWF-bound FVIII, and/orstabilizing the FVIII heavy chain and light chain interactions.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, if an amino acid in apolypeptide is replaced with another amino acid from the same side chainfamily, the substitution is considered to be conservative. In anotherembodiment, a string of amino acids can be conservatively replaced witha structurally similar string that differs in order and/or compositionof side chain family members.

As known in the art, “sequence identity” between two polypeptides isdetermined by comparing the amino acid sequence of one polypeptide tothe sequence of a second polypeptide. When discussed herein, whether anyparticular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 99%, or 100% identical to another polypeptide can bedetermined using methods and computer programs/software known in the artsuch as, but not limited to, the BESTFIT program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences. When using BESTFIT or anyother sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full-lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

As used herein, an “amino acid corresponding to” or an “equivalent aminoacid” in a VWF sequence or a FVIII protein sequence is identified byalignment to maximize the identity or similarity between a first VWF orFVIII sequence and a second VWF or FVIII sequence. The number used toidentify an equivalent amino acid in a second VWF or FVIII sequence isbased on the number used to identify the corresponding amino acid in thefirst VWF or FVIII sequence.

As used herein, the term “insertion site” refers to a position in aFVIII polypeptide, or fragment, variant, or derivative thereof, which isimmediately upstream of the position at which a heterologous moiety canbe inserted. An “insertion site” is specified as a number, the numberbeing the number of the amino acid in mature native FVIII (SEQ ID NO:4)to which the insertion site corresponds, which is immediately N-terminalto the position of the insertion. For example, the phrase “a3 comprisesan XTEN at an insertion site which corresponds to amino acid 1656 of SEQID NO: 4” indicates that the heterologous moiety is located between twoamino acids corresponding to amino acid 1656 and amino acid 1657 of SEQID NO: 4.

The phrase “immediately downstream of an amino acid” as used hereinrefers to position right next to the terminal carboxyl group of theamino acid. Similarly, the phrase “immediately upstream of an aminoacid” refers to the position right next to the terminal amine group ofthe amino acid. Therefore, the phrase “between two amino acids of aninsertion site” as used herein refers to a position in which an XTEN orany other polypeptide is inserted between two adjacent amino acids.Thus, the phrases “inserted immediately downstream of an amino acid” and“inserted between two amino acids of an insertion site” are usedsynonymously with “inserted at an insertion site.”

The terms “inserted,” “is inserted,” “inserted into” or grammaticallyrelated terms, as used herein refers to the position of an XTEN in achimeric polypeptide relative to the analogous position in native maturehuman FVIII. As used herein the terms refer to the characteristics ofthe recombinant FVIII polypeptide relative to native mature human FVIII,and do not indicate, imply or infer any methods or process by which thechimeric polypeptide was made. For example, in reference to a chimericpolypeptide provided herein, the phrase “an XTEN is inserted intoimmediately downstream of residue 745 of the FVIII polypeptide” meansthat the chimeric polypeptide comprises an XTEN immediately downstreamof an amino acid which corresponds to amino acid 745 in native maturehuman FVIII, e.g., bounded by amino acids corresponding to amino acids745 and 746 of native mature human FVIII.

A “fusion” or “chimeric” protein comprises a first amino acid sequencelinked to a second amino acid sequence with which it is not naturallylinked in nature. The amino acid sequences which normally exist inseparate proteins can be brought together in the fusion polypeptide, orthe amino acid sequences which normally exist in the same protein can beplaced in a new arrangement in the fusion polypeptide, e.g., fusion of aFactor VIII domain of the invention with an Ig Fc domain. A fusionprotein is created, for example, by chemical synthesis, or by creatingand translating a polynucleotide in which the peptide regions areencoded in the desired relationship. A chimeric protein can furthercomprises a second amino acid sequence associated with the first aminoacid sequence by a covalent, non-peptide bond or a non-covalent bond.

As used herein, the term “half-life” refers to a biological half-life ofa particular polypeptide in vivo. Half-life may be represented by thetime required for half the quantity administered to a subject to becleared from the circulation and/or other tissues in the animal. When aclearance curve of a given polypeptide is constructed as a function oftime, the curve is usually biphasic with a rapid α-phase and longerβ-phase. The α-phase typically represents an equilibration of theadministered Fc polypeptide between the intra- and extra-vascular spaceand is, in part, determined by the size of the polypeptide. The β-phasetypically represents the catabolism of the polypeptide in theintravascular space. In some embodiments, FVIII and chimeric proteinscomprising FVIII are monophasic, and thus do not have an alpha phase,but just the single beta phase. Therefore, in certain embodiments, theterm half-life as used herein refers to the half-life of the polypeptidein the β-phase. The typical β-phase half-life of a human antibody inhumans is 21 days.

The term “linked” as used herein refers to a first amino acid sequenceor nucleotide sequence covalently or non-covalently joined to a secondamino acid sequence or nucleotide sequence, respectively. The firstamino acid or nucleotide sequence can be directly joined or juxtaposedto the second amino acid or nucleotide sequence or alternatively anintervening sequence can covalently join the first sequence to thesecond sequence. The term “linked” means not only a fusion of a firstamino acid sequence to a second amino acid sequence at the C-terminus orthe N-terminus, but also includes insertion of the whole first aminoacid sequence (or the second amino acid sequence) into any two aminoacids in the second amino acid sequence (or the first amino acidsequence, respectively). In one embodiment, the first amino acidsequence can be linked to a second amino acid sequence by a peptide bondor a linker. The first nucleotide sequence can be linked to a secondnucleotide sequence by a phosphodiester bond or a linker. The linker canbe a peptide or a polypeptide (for polypeptide chains) or a nucleotideor a nucleotide chain (for nucleotide chains) or any chemical moiety(for both polypeptide and polynucleotide chains). The term “linked” isalso indicated by a hyphen (-).

As used herein the term “associated with” refers to a covalent ornon-covalent bond formed between a first amino acid chain and a secondamino acid chain. In one embodiment, the term “associated with” means acovalent, non-peptide bond or a non-covalent bond. This association canbe indicated by a colon, i.e., (:). In another embodiment, it means acovalent bond except a peptide bond. For example, the amino acidcysteine comprises a thiol group that can form a disulfide bond orbridge with a thiol group on a second cysteine residue. In mostnaturally occurring IgG molecules, the CH1 and CL regions are associatedby a disulfide bond and the two heavy chains are associated by twodisulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).Examples of covalent bonds include, but are not limited to, a peptidebond, a metal bond, a hydrogen bond, a disulfide bond, a sigma bond, api bond, a delta bond, a glycosidic bond, an agnostic bond, a bent bond,a dipolar bond, a Pi backbond, a double bond, a triple bond, a quadruplebond, a quintuple bond, a sextuple bond, conjugation, hyperconjugation,aromaticity, hapticity, or antibonding. Non-limiting examples ofnon-covalent bond include an ionic bond (e.g., cation-pi bond or saltbond), a metal bond, an hydrogen bond (e.g., dihydrogen bond, dihydrogencomplex, low-barrier hydrogen bond, or symmetric hydrogen bond), van derWalls force, London dispersion force, a mechanical bond, a halogen bond,aurophilicity, intercalation, stacking, entropic force, or chemicalpolarity.

The term “monomer-dimer hybrid” used herein refers to a chimeric proteincomprising a first polypeptide chain and a second polypeptide chain,which are associated with each other by a disulfide bond, wherein thefirst chain comprises a clotting factor, e.g., Factor VIII, and a firstFc region and the second chain comprises, consists essentially of, orconsists of a second Fc region without the clotting factor. Themonomer-dimer hybrid construct thus is a hybrid comprising a monomeraspect having only one clotting factor and a dimer aspect having two Fcregions.

As used herein, the term “cleavage site” or “enzymatic cleavage site”refers to a site recognized by an enzyme. Certain enzymatic cleavagesites comprise an intracellular processing site. In one embodiment, apolypeptide has an enzymatic cleavage site cleaved by an enzyme that isactivated during the clotting cascade, such that cleavage of such sitesoccurs at the site of clot formation. Exemplary such sites include,e.g., those recognized by thrombin, Factor XIa or Factor Xa. ExemplaryFXIa cleavage sites include, e.g., TQSFNDFTR (SEQ ID NO: 45) andSVSQTSKLTR (SEQ ID NO: 46). Exemplary thrombin cleavage sites include,e.g., DFLAEGGGVR (SEQ ID NO: 47), TTKIKPR (SEQ ID NO: 48), LVPRG (SEQ IDNO: 49) and ALRPR (amino acids 1 to 5 of SEQ ID NO: 50). Other enzymaticcleavage sites are known in the art.

As used herein, the term “processing site” or “intracellular processingsite” refers to a type of enzymatic cleavage site in a polypeptide whichis a target for enzymes that function after translation of thepolypeptide. In one embodiment, such enzymes function during transportfrom the Golgi lumen to the trans-Golgi compartment. Intracellularprocessing enzymes cleave polypeptides prior to secretion of the proteinfrom the cell. Examples of such processing sites include, e.g., thosetargeted by the PACE/furin (where PACE is an acronym for Paired basicAmino acid Cleaving Enzyme) family of endopeptidases. These enzymes arelocalized to the Golgi membrane and cleave proteins on thecarboxyterminal side of the sequence motif Arg-[any residue]-(Lys orArg)-Arg. As used herein the “furin” family of enzymes includes, e.g.,PCSK1 (also known as PC1/Pc3), PCSK2 (also known as PC2), PCSK3 (alsoknown as furin or PACE), PCSK4 (also known as PC4), PCSK5 (also known asPC5 or PC6), PCSK6 (also known as PACE4), or PCSK7 (also known asPC7/LPC, PC8, or SPC7). Other processing sites are known in the art.

In constructs that include more than one processing or cleavage site, itwill be understood that such sites may be the same or different.

The term “Furin” refers to the enzymes corresponding to EC No.3.4.21.75. Furin is subtilisin-like proprotein convertase, which is alsoknown as PACE (Paired basic Amino acid Cleaving Enzyme). Furin deletessections of inactive precursor proteins to convert them intobiologically active proteins. During its intracellular transport,pro-peptide of VWF can be cleaved from mature VWF molecule by a Furinenzyme. In some embodiments, Furin cleaves the D1D2 from the D′D3 ofVWF. In other embodiments, a nucleotide sequence encoding Furin can beexpressed together with the nucleotide sequence encoding a VWF fragmentso that D1D2 domains can be cleaved off intracellularly by Furin.

In constructs that include more than one processing or cleavage site, itwill be understood that such sites may be the same or different.

A “processable linker” as used herein refers to a linker comprising atleast one intracellular processing site, which is described elsewhereherein.

Hemostatic disorder, as used herein, means a genetically inherited oracquired condition characterized by a tendency to hemorrhage, eitherspontaneously or as a result of trauma, due to an impaired ability orinability to form a fibrin clot. Examples of such disorders include thehemophilias. The three main forms are hemophilia A (factor VIIIdeficiency), hemophilia B (factor IX deficiency or “Christmas disease”)and hemophilia C (factor XI deficiency, mild bleeding tendency). Otherhemostatic disorders include, e.g., Von Willebrand disease, Factor XIdeficiency (PTA deficiency), Factor XII deficiency, deficiencies orstructural abnormalities in fibrinogen, prothrombin, Factor V, FactorVII, Factor X or factor XIII, Bernard-Soulier syndrome, which is adefect or deficiency in GPIb. GPIb, the receptor for VWF, can bedefective and lead to lack of primary clot formation (primaryhemostasis) and increased bleeding tendency), and thrombasthenia ofGlanzman and Naegeli (Glanzmann thrombasthenia). In liver failure (acuteand chronic forms), there is insufficient production of coagulationfactors by the liver; this may increase bleeding risk.

The chimeric molecules of the invention can be used prophylactically. Asused herein the term “prophylactic treatment” refers to theadministration of a molecule prior to a bleeding episode. In oneembodiment, the subject in need of a general hemostatic agent isundergoing, or is about to undergo, surgery. The chimeric protein of theinvention can be administered prior to or after surgery as aprophylactic. The chimeric protein of the invention can be administeredduring or after surgery to control an acute bleeding episode. Thesurgery can include, but is not limited to, liver transplantation, liverresection, dental procedures, or stem cell transplantation.

The chimeric protein of the invention is also used for on-demandtreatment. The term “on-demand treatment” refers to the administrationof a chimeric molecule in response to symptoms of a bleeding episode orbefore an activity that may cause bleeding. In one aspect, the on-demandtreatment can be given to a subject when bleeding starts, such as afteran injury, or when bleeding is expected, such as before surgery. Inanother aspect, the on-demand treatment can be given prior to activitiesthat increase the risk of bleeding, such as contact sports.

As used herein the term “acute bleeding” refers to a bleeding episoderegardless of the underlying cause. For example, a subject may havetrauma, uremia, a hereditary bleeding disorder (e.g., factor VIIdeficiency) a platelet disorder, or resistance owing to the developmentof antibodies to clotting factors.

Treat, treatment, treating, as used herein refers to, e.g., thereduction in severity of a disease or condition; the reduction in theduration of a disease course; the amelioration of one or more symptomsassociated with a disease or condition; the provision of beneficialeffects to a subject with a disease or condition, without necessarilycuring the disease or condition, or the prophylaxis of one or moresymptoms associated with a disease or condition. In one embodiment, theterm “treating” or “treatment” means maintaining a FVIII trough level atleast about 1 IU/dL, 2 IU/dL, 3 IU/dL, 4 IU/dL, 5 IU/dL, 6 IU/dL, 7IU/dL, 8 IU/dL, 9 IU/dL, 10 IU/dL, 11 IU/dL, 12 IU/dL, 13 IU/dL, 14IU/dL, 15 IU/dL, 16 IU/dL, 17 IU/dL, 18 IU/dL, 19 IU/dL, or 20 IU/dL ina subject by administering a chimeric protein or a VWF fragment of theinvention. In another embodiment, treating or treatment meansmaintaining a FVIII trough level between about 1 and about 20 IU/dL,about 2 and about 20 IU/dL, about 3 and about 20 IU/dL, about 4 andabout 20 IU/dL, about 5 and about 20 IU/dL, about 6 and about 20 IU/dL,about 7 and about 20 IU/dL, about 8 and about 20 IU/dL, about 9 andabout 20 IU/dL, or about 10 and about 20 IU/dL. Treatment or treating ofa disease or condition can also include maintaining FVIII activity in asubject at a level comparable to at least about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% ofthe FVIII activity in a non-hemophiliac subject. The minimum troughlevel required for treatment can be measured by one or more knownmethods and can be adjusted (increased or decreased) for each person.

Chimeric Proteins

The present invention is directed to extending the half-life of a FactorVIII protein using a VWF fragment and an XTEN sequence by preventing orinhibiting a FVIII half-life limiting factor, i.e., endogenous VWF, fromassociating with the FVIII protein. Endogenous VWF associates with about95% to about 98% of FVIII in non-covalent complexes. While endogenousVWF is a FVIII half-life limiting factor, endogenous VWF bound to aFVIII protein is also known to protect FVIII in various ways. Forexample, full length VWF (as a multimer having about 250 kDa) canprotect FVIII from protease cleavage and FVIII activation, stabilize theFVIII heavy chain and/or light chain, and prevent clearance of FVIII byscavenger receptors. But, at the same time, endogenous VWF limits theFVIII half-life by preventing pinocytosis and by clearing FVIII-VWFcomplex from the system through the VWF clearance pathway. It isbelieved, while not bound by a theory, that endogenous VWF is ahalf-life limiting factor that prevents the half-life of a FVIII proteinfused to a half-life extender from being longer than about two-fold thatof wild-type FVIII. Therefore, the present invention is directed topreventing or inhibiting interaction between endogenous VWF and a FVIIIprotein using a VWF fragment, thereby increasing a half-life of theFVIII protein by using an XTEN sequence alone or an XTEN sequence incombination with an Ig constant region or a portion thereof. The XTENsequence can be linked to the FVIII protein or the VWF fragment. TheFVIII protein associated with the VWF fragment is thus cleared from thecirculation more slowly by one or more VWF clearance receptors and thencan have the full half-life extension of the XTEN sequence or the XTENsequence in combination of the Ig constant region, as compared to wildtype FVIII or a FVIII protein without the VWF fragment.

In one embodiment, a VWF fragment is associated (or linked) with theFVIII protein by a covalent or a non-covalent bond. In some instances,however, the physical blockage or chemical association (e.g.,non-covalent bonding) between the VWF fragment and the FVIII protein maynot be strong enough to provide a stable complex comprising the FVIIIprotein and the VWF fragment in the presence of endogenous VWF. Forexample, a VWF fragment forming a non-covalent bond with a FVIII proteinwithout any other connections may readily be dissociated in vivo fromthe FVIII protein in the presence of endogenous VWF, replacing the VWFfragment (e.g., recombinant VWF, i.e., rVWF) with endogenous VWF.Therefore, the FVIII protein non-covalently bound to endogenous VWFwould undergo the VWF clearance pathway and be readily cleared from thesystem. In order to prevent the dissociation of the VWF fragment withthe FVIII protein, in some embodiments, the association or linkagebetween the FVIII protein and the VWF fragment is a covalent bond, e.g.,a peptide bond, one or more amino acids, or a disulfide bond. In certainembodiments, the association (i.e., linkage) between the adjunct moietyand the FVIII protein is a peptide bond or a linker between the FVIIIprotein and the VWF fragment (“FVIII/VWF linker”). Non-limiting examplesof the linker are described elsewhere herein. In some embodiments, theVWF fragment is a polypeptide comprising, consisting essentially of, orconsisting of at least about 10, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2500, 3000, or 4000 amino acids. Non-limiting examples of the VWFfragment are described elsewhere herein.

In certain embodiments, the VWF fragment chemically (e.g.,non-covalently) binds to or physically blocks one or more VWF bindingsites on a FVIII protein. The VWF binding site on a FVIII protein islocated within the A3 domain or the C2 domain of the FVIII protein. Instill other embodiments, the VWF binding site on a FVIII protein islocated within the A3 domain and C2 domain. For example, the VWF bindingsite on a FVIII protein can correspond to amino acids 1669 to 1689and/or 2303 to 2332 of SEQ ID NO: 4 [full-length mature FVIII].

The invention also provides a chimeric protein (comprising a FVIIIprotein and a VWF fragment) further comprising one or more XTENsequences, which provide additional half-life extension properties. Theone or more XTEN sequences can be inserted within the FVIII protein orthe VWF fragment or linked to the N-terminus or the C-terminus of theFVIII protein or the VWF fragment. The invention also includes a FVIIIprotein linked to an XTEN sequence (a first half-life extending moiety)and an Ig constant region or a portion thereof (a second half-lifeextending moiety) so that the two half-life extending moieties extendthe half-life of the FVIII protein through two different mechanisms.

In some embodiments, a chimeric protein comprises a FVIII protein linkedto a first Ig constant region or a portion thereof (e.g., a first FcRnbinding partner), a VWF fragment linked to a second Ig constant regionor a portion thereof (e.g., a second FcRn binding partner), and one ormore XTEN sequences inserted or linked to the FVIII protein or the VWFfragment, wherein the VWF fragment prevents the FVIII half-life limitingfactor (e.g., endogenous VWF) from binding to the FVIII protein, whereinthe first and second Ig constant regions or portions thereof forms acovalent bond, e.g., a disulfide bond, and the one or more XTENsequences extends the half-life of the FVIII protein.

In certain embodiments, a chimeric protein of the invention comprises aFVIII protein linked to a VWF fragment by an optional linker (i.e.,FVIII/VWF linker) and one or more XTEN sequences inserted or linked tothe FVIII protein or the VWF fragment, wherein the VWF fragment preventsthe FVIII half-life limiting factor (e.g., endogenous VWF) from bindingto the FVIII protein and the one or more XTEN sequences extends thehalf-life of the FVIII protein. In one aspect, the optional linker(FVIII/VWF linker) comprises a sortase recognition motif. In anotheraspect, the optional linker (FVIII/VWF linker) comprises a cleavablesite. Examples of the cleavage linker (i.e., linker containing one ormore cleavage site) are described elsewhere herein.

The chimeric protein of the present invention includes, but is notlimited to:

(1) a VWF fragment comprising a D′ domain and a D3 domain, an XTENsequence, and FVIII, wherein the XTEN sequence is linked to the VWFfragment;(2) a FVIII protein, an XTEN sequence, and an Ig constant region or aportion thereof, wherein the FVIII protein is linked to an XTEN sequenceand the Ig constant region or a portion thereof, or(3) a FVIII protein, an XTEN sequence, and a VWF fragment, wherein theXTEN sequence is linked to the FVIII protein at the C-terminus orN-terminus or inserted immediately downstream of one or more amino acids(e.g., one or more XTEN insertion sites) of FVIII, and the VWF fragmentand the FVIII protein are associated with each other.

(1) Von Willebrand Factor (VWF) Fragment Linked to XTEN, and FVIII

The present invention is directed to a chimeric protein comprising (i) aVWF fragment comprising a D′ domain and a D3 domain of VWF, (ii) an XTENsequence, and (iii) a FVIII protein, wherein (i), (ii), and (iii) arelinked to or associated with each other. The VWF fragment linked to theXTEN sequence, as a part of a chimeric protein in the present invention,associates with the FVIII protein, thus preventing or inhibitinginteraction between endogenous VWF and the FVIII protein. In certainembodiments, the VWF fragment, which is capable of preventing orinhibiting binding of the FVIII protein with endogenous VWF, can at thesame time have at least one VWF-like FVIII protecting property. Examplesof the VWF-like FVIII protecting properties include, but are not limitedto, protecting FVIII from protease cleavage and FVIII activation,stabilizing the FVIII heavy chain and/or light chain, and preventingclearance of FVIII by scavenger receptors. As a result, the VWF fragmentcan prevent clearance of the FVIII protein through the VWF clearancepathway, thus reducing clearance of FVIII from the circulatory system.In some embodiments, the VWF fragments of the present invention bind toor are associated with a FVIII protein and/or physically or chemicallyblock the VWF binding site on the FVIII protein. The FVIII proteinassociated with the VWF fragment is thus cleared from the circulationmore slowly, as compared to wild type FVIII or FVIII not associated withthe VWF fragment.

In one embodiment, the invention is directed to a chimeric proteincomprising (i) a VWF fragment comprising the D′ domain and the D3 domainof VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein theXTEN sequence is linked to the VWF fragment (e.g., (a1) V-X or (a2) X-V,wherein V comprises a VWF fragment and X comprises an XTEN sequence),and the VWF fragment is linked to or associated with the FVIII protein.In another embodiment, the VWF fragment and the XTEN sequence can belinked by a linker (e.g., (a3) V-L-X or (a4) X-L-V) or a peptide bond.The linker can be a cleavable linker, e.g., a thrombin cleavable linker,which can be cleaved at the site of coagulation. In other embodiments,the VWF fragment, the XTEN sequence, and the FVIII protein are placed ina single polypeptide chain. In still other embodiments, the chimericprotein comprises two polypeptide chains, a first chain comprising theVWF fragment and the XTEN sequence and a second chain comprising theFVIII protein. In yet other embodiments, the chimeric protein comprisesthree polypeptide chains, a first chain comprising the VWF fragment andthe XTEN sequence, a second chain comprising a light chain of FVIII anda third chain comprising a heavy chain of FVIII, wherein the first chainand the second chain are associated with each other (e.g., covalentbond, e.g., disulfide bond), and the second chain and the third chainare associated with each other (e.g., metal bond). In still otherembodiments, the XTEN sequence can be linked to the N-terminus or theC-terminus of the VWF fragment or inserted immediately downstream of oneor more amino acids in the VWF fragment.

In certain embodiments, a chimeric protein of the invention comprises aformula comprising:

V-X-FVIII,  (a)

FVIII-X-V,  (b)

V-X:FVIII,  (c)

X-V:FVIII,  (d)

FVIII:V-X,  (e)

FVIII:X-V, or  (f)

X-V-FVIII,  (a5)

wherein V comprises a VWF fragment,X comprises one or more XTEN sequences,FVIII comprises a FVIII protein;(-) represents a peptide bond or one or more amino acids; and(:) is a chemical association or a physical association. In oneembodiment, (:) represents a chemical association, e.g., at least onenon-peptide bond. In another embodiment, the chemical association, i.e.,(:) is a covalent bond. In other embodiments, the chemical association,i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, ahydrophobic interaction, a hydrophilic interaction, a Van der Waalsinteraction, or a hydrogen bond. In other embodiments, (:) is anon-peptide covalent bond. In still other embodiments, (:) is a peptidebond. In yet other embodiments, (:) represents a physical associationbetween two sequences, wherein a portion of a first sequence is in closeproximity to a second sequence such that the first sequence shields orblocks a portion of the second sequence from interacting with anothermoiety, and further that this physical association is maintained withoutallowing the second sequence to interact with other moieties. Theorientation of the polypeptide formulas herein is listed from N-terminus(left) to C-terminus (right). For example, formula V-X-FVIII meansformula NH2-V-X-FVIII-COOH. In one embodiment, the formulas describedherein can comprise any additional sequences between the two moieties.For example, formula V-X-FVIII can further comprise any sequences at theN-terminus of V between V and X, between X and FVIII, or at theC-terminus of FVIII unless otherwise specified. In another embodiment,the hyphen (-) indicates a peptide bond.

In other embodiments, a chimeric protein of the invention comprises aformula comprising:

V(X1)-X2-FVIII,  (a)

FVIII-X2-V(X1),  (b)

V(X1):FVIII,  (c)

FVIII:V(X1), or  (d)

X2-V(X1)-FVIII,  (a5)

wherein V(X1) comprises a VWF fragment and a first XTEN sequence (X1),wherein the XTEN sequence is inserted immediately downstream of one ormore amino acids in the VWF fragment,X2 comprises one or more optional XTEN sequences, FVIII comprises aFVIII protein;(-) is a peptide bond or one or more amino acids; and(:) is a chemical association or a physical association.

In some embodiments, a chimeric protein comprises (i) a VWF fragmentcomprising a D′ domain and a D3 domain of VWF, (ii) an XTEN sequence,(iii) a FVIII protein, (iv) a first optional linker, and (v) a secondoptional linker, wherein the XTEN sequence is linked to the VWF fragmentand/or to the FVIII protein by the linker. In certain embodiments, achimeric protein comprises a formula comprising:

V-L1-X-L2-FVIII,  (b1)

FVIII-L2-X-L1-V,  (b2)

V-L1-X:FVIII,  (b3)

X-L1-V:FVIII,  (b4)

FVIII:V-L1-X,  (b5)

FVIII:X-L1-V,  (b6)

X-L1-V-L2-FVIII, or  (b7)

FVIII-L2-V-L1-X,  (b8)

wherein V comprises a VWF fragment,X comprises one or more XTEN sequences,FVIII comprises a FVIII protein,L1 comprises a first optional linker, e.g., a first cleavable linker,L2 comprises a second optional linker, e.g., a second cleavable linkeror an optional processable linker;(-) is a peptide bond or one or amino acids; and(:) is a chemical association or a physical association. In oneembodiment, (:) represents a chemical association, e.g., at least onenon-peptide bond. In another embodiment, the chemical association, i.e.,(:) is a covalent bond. In other embodiments, the chemical association,i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, ahydrophobic interaction, a hydrophilic interaction, a Van der Waalsinteraction, or a hydrogen bond. In other embodiments, (:) is anon-peptide covalent bond. In still other embodiments, (:) is a peptidebond. In yet other embodiments, (:) represents a physical associationbetween two sequences, wherein a portion of a first sequence is in closeproximity to a second sequence such that the first sequence shields orblocks a portion of the second sequence from interacting with anothermoiety, and further that this physical association is maintained withoutallowing the second sequence to interact with other moieties. Theorientation of the polypeptide formulas herein is listed from N-terminus(left) to C-terminus (right). For example, formula (b1) V-L1-X-L2-FVIIImeans formula NH2-V-L1-X-L2-FVIII-COOH. In one embodiment, the formulasdescribed herein can comprise any additional sequences between the twomoieties. In another embodiment, the hyphen (-) indicates a peptidebond.

Another aspect of the present invention is to provide a FVIII chimericprotein having reduced or no interactions with a FVIII half-lifelimiting factor, e.g., endogenous VWF, and at the same time maximizingthe half-life of the FVIII protein using an XTEN sequence (a firsthalf-life extender) in combination with a second half-life extender or amoiety providing a covalent bond between the FVIII protein and the VWFfragment, e.g., an Ig constant region or a portion thereof. In oneembodiment, a chimeric protein of the invention comprises (i) a VWFfragment comprising a D′ domain and a D3 domain of VWF, (ii) an XTENsequence, (iii) a FVIII protein, and (iv) an Ig constant region or aportion thereof (also referred to herein as F), wherein (1) the VWFfragment is linked to the XTEN sequence by an optional linker, e.g., acleavable linker, (2) the VWF fragment is associated with or linked tothe FVIII protein by an additional optional linker, e.g., a cleavablelinker, and (3) the Ig constant region or a portion thereof is linked tothe VWF fragment, the XTEN sequence, or the FVIII protein. In anotherembodiment, a chimeric protein of the invention comprises (i) a VWFfragment comprising a D′ domain and a D3 domain of VWF, (ii) an XTENsequence, (iii) a FVIII protein, (iv) an Ig constant region or a portionthereof (F1 or a first Ig constant region or a portion thereof), and (v)an additional Ig constant region or a portion thereof (F2 or a second Igconstant region or a portion thereof), wherein (1) the VWF fragment islinked to the XTEN sequence by an optional linker, e.g., a cleavablelinker, (2) the XTEN sequence or the VWF fragment is linked to the Igconstant region or a portion thereof, (3) the FVIII is linked to theadditional Ig constant region or a portion thereof, and (4) the Igconstant region or a portion thereof is associated with or linked to theadditional Ig constant region or a portion thereof. In one embodiment,the association or linkage between the two Ig constant regions or aportion thereof is a covalent bond, e.g., a disulfide bond. In anotherembodiment, the association or linkage between the two Ig constantregions or a portion thereof is a processable linker, wherein theprocessable linker is intracellularly processed by a protease. Forexample, the chimeric protein comprises a formula comprising:

V-L2-X-L1-F1:FVIII-L3-F2;  (g)

V-L2-X-L1-F1:F2-L3-FVIII;  (h)

F-L1-X-L2-V: FVIII-L3-F2;  (i)

F-L1-X-L2-V:F2-L3-FVIII;  (j)

V-L2-X-L1-F1-L4-FVIII-L3-F2;  (k)

F2-L3-FVIII-L4-F1-L1-X-L2-V;  (l)

FVIII-L2-F2-L4-V-L2-X-L1-F1; or  (m)

F1-L1-X-L2-V-L4-F2-L2-FVIII,  (n)

wherein V comprises a VWF fragment,each of L1 and L3 comprises an optional linker,L2 comprises an optional linker, e.g., a cleavable linker,L4 is an optional linker, e.g., a processable linker,FVIII comprises a FVIII protein,X comprises one or more XTEN sequences,F1 comprises an optional Ig constant region or a portion thereof,F2 comprises an optional additional Ig constant region or a portionthereof;(-) is a peptide bond or one or more amino acids; and(:) is a chemical association or a physical association.

In some embodiments, the FVIII protein in any constructs or formulasdisclosed herein can further comprises at least one, at least two, atleast three, at least four, at least five, or at least six XTENsequences, each of the XTEN sequences inserted immediately downstream ofone or more amino acids in the FVIII protein or linked to the N-terminusor the C-terminus of the FVIII protein. Non-limiting examples of theXTEN insertion sites are disclosed elsewhere herein.

In one embodiment, (:) represents a chemical association, e.g., at leastone non-peptide bond. In another embodiment, the chemical association,i.e., (:) is a covalent bond. In other embodiments, the chemicalassociation, i.e., (:) is a non-covalent interaction, e.g., an ionicinteraction, a hydrophobic interaction, a hydrophilic interaction, a Vander Waals interaction, or a hydrogen bond. In other embodiments, (:) isa non-peptide covalent bond. In still other embodiments, (:) is apeptide bond. In yet other embodiments, (:) represents a physicalassociation between two sequences, wherein a portion of a first sequenceis in close proximity to a second sequence such that the first sequenceshields or blocks a portion of the second sequence from interacting withanother moiety, and further that this physical association is maintainedwithout allowing the second sequence to interact with other moieties.The orientation of the polypeptide formulas herein is listed fromN-terminus (left) to C-terminus (right). For example, formula (n)F1-L1-X-L2-V-L4-F2-L2-FVIII means formulaNH2-F1-L1-X-L2-V-L4-F2-L2-FVIII-COOH. In one embodiment, the formulasdescribed herein can comprise any additional sequences between the twomoieties. In another embodiment, the hyphen (-) indicates a peptidebond.

In one embodiment, either or both of the Ig constant region or a portionthereof (sometimes indicated herein by “F” or “F1”) and the additionalIg constant region or a portion thereof (sometimes indicated herein by“F2”) linked to the VWF fragment or the FVIII protein can extend thehalf-life of the VWF fragment, the FVIII protein, or both. In anotherembodiment, a pair of the Ig constant region or a portion thereof(sometimes indicated herein by “F” or “F1”) and the additional Igconstant region or a portion thereof (sometimes indicated herein by“F2”), each of which are linked to the VWF fragment and the FVIIIprotein, provides a bond stronger than the non-covalent bond between theFVIII protein and the VWF fragment, i.e., a covalent bond, e.g., adisulfide bond, thereby preventing endogenous VWF from replacing the VWFfragment in vivo. F1 or F2 can comprise an Fc region or an FcRn bindingpartner. In other embodiments, either or both of F1 and F2 linked to theVWF fragment and/or the FVIII protein form a covalent bond (e.g., adisulfide bond) between F1 and F2, thereby placing the VWF fragment andthe FVIII protein in close proximity to prevent interaction of the FVIIIprotein with the VWF fragment. In some embodiments, F1 and F2 areidentical or different. Non-limiting examples of F1 and F2 can beselected from the group consisting of a CH1 domain, a CH2 domain, a CH3domain, a CH4 domain, a hinge domain, any functional fragments,derivatives, or analogs thereof, and two or more combinations thereof.In one embodiment, F1, F2, or both comprise at least one CH1 domain, atleast one CH2 domain, at least one CH3 domain, at least one CH4 domain,or the functional fragments, derivatives, or analogs thereof. In anotherembodiment, F1, F2, or both comprise at least one hinge domain orportion thereof and at least one CH2 domain or portion thereof (e.g., inthe hinge-CH2 orientation). In other embodiments, F1, F2, or bothcomprise at least one CH2 domain or portion thereof and at least one CH3domain or portion thereof (e.g., in the CH2-CH3 orientation.) Examplesof the combination include, but are not limited to, a CH2 domain, a CH3domain, and a hinge domain, which are also known as an Fc region (or Fcdomain), e.g., a first Fc region or a first FcRn binding partner for F1and a second Fc region or a second FcRn binding partner for F2. In otherembodiments, F1 is linked to the VWF fragment by a linker, and/or F2 islinked to the FVIII protein by a linker. In some embodiments, F1 and/orF2 comprises, consisting essentially of, or consisting of a hingeregion. Additional non-limiting examples of the Fc regions or the FcRnbinding partners are described elsewhere herein.

In certain embodiments, a chimeric protein of the invention comprisestwo polypeptide chains, a first polypeptide chain comprising, consistingessentially of, or consisting of a VWF fragment comprising a D′ domainand a D3 domain, an XTEN sequence, a first Ig constant region or aportion thereof (e.g., a first Fc region), and an optional linkerbetween the VWF fragment and the XTEN sequence or the XTEN sequence orthe first Ig constant region or a portion thereof and a secondpolypeptide chain comprising, consisting essentially of, or consistingof a FVIII protein and a second Ig constant region or a portion thereof(e.g., a second Fc region). The linker between the VWF fragment and thefirst Ig constant region or a portion thereof can be a cleavable linker,e.g., a thrombin cleavable linker, which can be cleaved at the site ofcoagulation. In some embodiments, the first polypeptide chain and thesecond polypeptide chain are associated with each other. The associationbetween the first chain and the second chain prevents replacement of thefirst chain comprising the VWF fragment with endogenous VWF in vivo. Inone embodiment, the association between the first chain and the secondchain can be a covalent bond. In a particular embodiment, the covalentbond is a disulfide bond. In some embodiments, the FVIII protein in thesecond chain further comprises one or more XTEN sequences linked to theC-terminus or N-terminus of the FVIII protein or inserted immediatelydownstream of one or more amino acids (e.g., at least one insertion sitedisclosed herein) in the FVIII protein. Non-limiting examples of theinsertion sites are described elsewhere herein.

In other embodiments, a chimeric protein of the invention comprisesthree polypeptide chains, wherein a first polypeptide chain comprises,consists essentially of, or consists of a heavy chain of a FVIIIprotein, a second polypeptide chain comprises, consists essentially of,or consists of a light chain of a FVIII protein fused to a first Igconstant region or a portion thereof (e.g., a first Fc region), and athird polypeptide chain comprises, consists essentially of, or consistsof a VWF fragment comprising a D′ domain and a D3 domain, an XTENsequence, a second Ig constant region or a portion thereof (e.g., asecond Fc region), and an optional linker between the XTEN sequence andthe second Ig constant region or a portion thereof or the VWF fragmentand the XTEN sequence. The linker in the third chain can be a cleavablelinker, which is cleaved at the site of coagulation, e.g., a thrombincleavage site. In some embodiments, the heavy chain FVIII or the lightchain FVIII is linked to one or more XTEN sequences, which can be linkedto the N-terminus, the C-terminus, or inserted within one or moreinsertion sites within the FVIII sequence. Non-limiting examples of theinsertion sites are disclosed elsewhere herein.

In yet other embodiments, a chimeric protein of the invention comprisestwo polypeptide chains, a first polypeptide chain comprising, consistingessentially of, or consisting of a heavy chain of a FVIII protein and asecond polypeptide chain comprising, consisting essentially of, orconsisting of a light chain of a FVIII protein, a first Ig constantregion or a portion thereof (e.g., a first Fc region), a first linker(e.g., a processable linker, which contains one or more proteasecleavage sites comprising one or more intracellular processing sites), aVWF fragment, a second linker (e.g., a thrombin cleavable linker), anXTEN sequence, and a second Ig constant region or a portion thereof(e.g., a second Fc region), wherein the light chain of the FVIII proteinis linked to the first Ig constant region or a portion thereof (e.g.,the first Fc region), which is further linked to the VWF fragment by thefirst linker, and wherein the VWF fragment is linked to the XTENsequence, which is further linked to the second Ig constant region or aportion thereof by the second linker. In certain embodiments, the firstlinker is a processable linker, and the second linker is a cleavablelinker. Upon expression, the chimeric protein can be processed by anintracellular processing enzyme, which cleaves the processable linker,and thus the chimeric protein can comprise, consists essentially of, orconsists of three polypeptide chains. In addition, the VWF fragment canbe cleaved off at the site of coagulation due to the cleavable linker.

In certain embodiments, a chimeric protein of the invention comprisesone polypeptide chain, which comprises a single chain FVIII protein, afirst Ig constant region or a portion thereof (e.g., a first Fc region),a first linker (e.g., a processable linker), a VWF fragment, an XTENsequence, a second linker (e.g., a thrombin cleavable linker), and asecond Ig constant region or a portion thereof (e.g., a second Fcregion), wherein the single chain FVIII protein is linked to the firstIg constant region or a portion thereof, which is also linked to the VWFfragment by the first linker, and the VWF fragment is linked to the XTENsequence, which is further linked to the second Ig constant region or aportion thereof. In one embodiment, the VWF fragment and the XTENsequence are linked by the second linker. In another embodiment, theXTEN sequence and the second Ig constant region or a portion thereof arelinked by the second linker. In other embodiments, the second chainfurther comprises a third linker. The single polypeptide chain can thuscomprise the VWF fragment linked to the XTEN sequence by the secondlinker and the XTEN linked to the second Ig constant region or a portionthereof by the third linker. The second linker and the third linker canbe identical or different. In one embodiment, the first linker is aprocessable linker. In another embodiment, the second linker or thethird linker is a cleavable linker comprising one or two cleavablesites. In a specific embodiment, the second linker is a thrombincleavable linker. The linkers useful in the invention are describedelsewhere herein.

(2) FVIII, XTEN, and Fe

A chimeric protein of the invention also comprises (i) a FVIII protein,(ii) an XTEN sequence (a first half-life extender), and (iii) an Igconstant region or a portion thereof (a second half-life extender), inwhich the XTEN sequence is linked to the FVIII protein by an optionallinker and the Ig constant region or a portion thereof by an additionaloptional linker. The XTEN sequence and the Ig constant region or aportion thereof can be used together to extend half-life of the FVIIIprotein. In one embodiment, the chimeric protein is a monomer. Inanother embodiment, the chimeric protein is a dimer (a homodimer or aheterodimer).

The present invention is also directed to a chimeric protein comprising(i) a FVIII protein, (ii) an XTEN sequence, (iii) an Ig constant regionor a portion thereof (i.e., a first Ig constant region or a portionthereof, “F,” or “F1”), and (iv) an additional Ig constant region or aportion thereof (i.e., a second Ig constant region or a portion thereofor “F2”). In one embodiment, the XTEN sequence is linked to the FVIIIprotein at the C-terminus or the N-terminus or inserted immediatelydownstream of one or more amino acids in the FVIII protein (e.g., one ormore XTEN insertion sites), the FVIII protein is linked to the first Igconstant region or a portion thereof, and the first Ig constant regionor a portion thereof and the second Ig constant region or a portionthereof are associated with or linked to each other by an optionallinker. In certain aspects, the chimeric protein is a monomer-dimerhybrid, which comprises a first polypeptide chain and a secondpolypeptide chain, wherein the first polypeptide chain comprises a FVIIIprotein, an XTEN sequence, and a first Ig constant region or a portionthereof, and the second polypeptide chain comprises, consistsessentially of, or consists of a second Ig constant region or a portionthereof without the FVIII protein and wherein the first chain and thesecond chain are associated with each other. The association between theIg constant region or a portion thereof (e.g., the first Fc region) andthe additional Ig constant region or a portion thereof (e.g., a secondFc region) is a chemical association or a physical association. Incertain embodiments, the chemical association is a covalent bond. Inother embodiments, the chemical association is a non-covalentinteraction, e.g., an ionic interaction, a hydrophobic interaction, ahydrophilic interaction, a Van der Waals interaction, or a hydrogenbond. In other embodiments, the association is a non-peptide covalentbond. In still other embodiments, the association is a peptide bond.

In other aspects, the chimeric protein is a single polypeptide chaincomprising a FVIII protein, an XTEN sequence, a first Ig constant regionor a portion thereof, a linker, e.g., a processable linker, and a secondIg constant region or a portion thereof, wherein the single polypeptidechain is processed after expression by an intracellular enzyme andbecomes two polypeptide chains.

In one embodiment, the Ig constant region or a portion thereof(sometimes indicated herein by “F” or “F1”) linked to the FVIII proteincan extend the half-life of the FVIII protein together with the XTENsequence. In another embodiment, the Ig constant region or a portionthereof (“F” or “F1”) is an Fc region or an FcRn binding partnerdescribed elsewhere herein.

In other embodiments, the additional Ig constant region or a portionthereof (sometimes indicated herein by “F2” or a second Ig constantregion or a portion thereof) associated with or linked to the first Igconstant region or a portion thereof can also extend the half-life ofthe FVIII protein. In other embodiments, the second Ig constant regionor a portion thereof (“F2”) together with the first Ig constant regionor a portion thereof and the XTEN sequence can extend the half-life ofthe FVIII protein. The additional Ig constant region or a portionthereof can be an Fc region or an FcRn binding partner describedelsewhere herein.

In certain embodiments, the second Ig constant region or a portionthereof associated with the first Ig constant region or a portionthereof is further linked to a VWF fragment described elsewhere hereinand an optional XTEN sequence.

In some embodiments, either or both of the Ig constant region or aportion thereof (“F” or “F1” or a first Ig constant region or a portionthereof) and an additional Ig constant region or a portion thereof(i.e., a second Ig constant region or a portion thereof or “F2”)(indicated in this paragraph as “the Ig constant regions or portionthereof”) can include, but not limited to, a CH1 domain, a CH2 domain, aCH3 domain, a CH4 domain, a hinge domain, any functional fragments,derivatives, or analogs thereof or two or more combinations thereof. Inone embodiment, the Ig constant region or a portion thereof comprises atleast one CH1 domain, at least one CH2 domain, at least one CH3 domain,at least one CH4 domain, or the functional fragments, derivatives, oranalogues thereof. In another embodiment, the Ig constant region or aportion thereof comprises at least one hinge domain or portion thereofand at least one CH2 domain or portion thereof (e.g., in the hinge-CH2orientation). In other embodiments, the Ig constant domain or portionthereof comprises at least one CH2 domain or portion thereof and atleast one CH3 domain or portion thereof (e.g., in the CF2-CH3orientation). Examples of the combination include, but are not limitedto, a CH2 domain, a CH3 domain, and a hinge domain, which are also knownas an Fc region (or Fc domain), e.g., first Fc region. Additionalexamples of the Ig constant regions or portion thereof are describedelsewhere herein.

The chimeric protein of the invention can have an extended half-life ofthe FVIII protein compared to wild-type FVIII. In one embodiment, thehalf-life of the FVIII protein is extended at least about 1.5 times, atleast about 2 times, at least about 2.5 times, at least about 3 times,at least about 4 times, at least about 5 times, at least about 6 times,at least about 7 times, at least about 8 times, at least about 9 times,at least about 10 times, at least about 11 times, or at least about 12times longer than the half-life of wild type FVIII. In anotherembodiment, the half-life of the FVIII protein is at least about 10hours, at least about 11 hours, at least about 12 hours, at least about13 hours, at least about 14 hours, at least about 15 hours, at leastabout 16 hours, at least about 17 hours, at least about 18 hours, atleast about 19 hours, at least about 20 hours, at least about 21 hours,at least about 22 hours, at least about 23 hours, at least about 24hours, at least about 36 hours, at least about 48 hours, at least about60 hours, at least about 72 hours, at least about 84 hours, at leastabout 96 hours, or at least about 108 hours.

(3) FVIII, XTEN, and VWF

In one aspect, a chimeric protein of the present invention comprises (i)a FVIII protein, (ii) an XTEN sequence, and (iii) a VWF fragmentcomprising a D′ domain and a D3 domain of VWF, wherein the FVIII proteinis linked to the XTEN sequence and wherein the FVIII protein isassociated with or linked to the VWF fragment. In one embodiment, theVWF fragment of the chimeric protein described herein is not capable ofbinding to a VWF clearance receptor. In another embodiment, the VWFfragment is capable of protecting the FVIII protein from one or moreprotease cleavages, protecting the FVIII protein from activation,stabilizing the heavy chain and/or the light chain of the FVIII protein,or preventing clearance of the FVIII protein by one or more scavengerreceptors. In other embodiments, the VWF fragment prevents or inhibitsbinding of endogenous VWF to the VWF binding site in the FVIII protein.The VWF binding site can be located in the A3 domain or the C2 domain ofthe FVIII protein or both the A3 domain and the C2 domain. In a specificembodiment, the VWF binding site comprises the amino acid sequencecorresponding to amino acids 1669 to 1689 and/or amino acids 2303 to2332 of SEQ ID NO: 2.

In another aspect, a chimeric protein comprises (i) a FVIII protein,(ii) an XTEN sequence, (iii) a VWF fragment, which comprises a D′ domainand a D3 domain of VWF, and (iv) an Ig constant region or a portionthereof, wherein the XTEN sequence is linked to the FVIII protein at theC-terminus or the N-terminus or inserted immediately downstream of oneor more amino acids (e.g., one or more XTEN insertion sites disclosedherein) in the FVIII protein, the VWF fragment is linked to orassociated with the FVIII protein or the XTEN sequence, and the Igconstant region or a portion thereof is linked to the FVIII protein, theXTEN sequence, the VWF fragment, or any combinations thereof. The Igconstant region or a portion thereof useful for chimeric proteins of theinvention is described elsewhere herein. In one embodiment, the Igconstant region or a portion thereof is capable of extending thehalf-life of a FVIII protein. In another embodiment, the Ig constantregion or a portion thereof comprises a first Fc region or a first FcRnbinding partner. In yet other embodiments, the Ig constant region or aportion thereof is linked to the FVIII protein by an optional linker. Instill other embodiments, the linker comprises a cleavable linker. Thechimeric protein can be a single polypeptide chain, i.e., a monomer(i.e., a single chain), containing (i), (ii), (iii), and (iv) or twochains containing a first chain comprising (i) and (ii) and a secondchain comprising (iii) and (iv). In other aspects, the chimeric proteinis a dimer (e.g., a homodimer or a heterodimer). In one embodiment, thechimeric protein comprises two chains, each comprising (i), (ii), (iii),and (iv).

In certain embodiments, a chimeric protein comprises (i) a FVIIIprotein, (ii) an XTEN sequence, (iii) a VWF fragment, which comprises aD′ domain and a D3 domain of VWF, (iv) an Ig constant region or aportion thereof (sometimes also indicated as “F,” “a first Ig constantregion or a portion thereof”, or “F2”), and (v) an additional Igconstant region or a portion thereof (sometimes also indicated as “F2”or “a second Ig constant region or a portion thereof”), wherein (1) theFVIII protein is linked to the XTEN sequence at the C-terminus orN-terminus of the FVIII protein or inserted immediately downstream ofone or more amino acids (e.g., one or more XTEN insertion sitesdisclosed herein) in the FVIII protein, (2) either the XTEN sequence orthe FVIII protein is linked to the Ig constant region or a portionthereof, (3) the VWF fragment is linked to the second Ig constant regionor a portion thereof, and (4) the Ig constant region or a portionthereof is associated with the second Ig constant region or a portionthereof. In one embodiment, the Ig constant region or a portion thereoflinked to the FVII protein or the XTEN sequence is further linked to theVWF fragment by a linker, e.g., a processable linker. In anotherembodiment, the additional Ig constant region or a portion thereofuseful for chimeric proteins of the invention can further be linked tothe FVIII protein or the Ig constant region or a portion thereof by anoptional linker, e.g., a processable linker. In some embodiments, a pairof the Ig constant region or a portion thereof and the additional Igconstant region or a portion thereof, each of which are linked to theVWF fragment and the FVIII protein, provides a bond stronger than thenon-covalent bond between the FVIII protein and the VWF fragment, i.e.,a covalent bond, e.g., a disulfide bond, thereby preventing endogenousVWF from replacing the VWF fragment in vivo. In other embodiments,either or both of the Ig constant region or a portion thereof and theadditional Ig constant region or a portion thereof are capable ofextending a half-life of the FVIII protein or the VWF fragment. In otherembodiments, the additional Ig constant region or a portion thereofcomprises a second Fc region or an FcRn binding partner. The Ig constantregion or a portion thereof and the additional Ig constant region or aportion thereof in the chimeric proteins are identical or different.

In certain embodiments, the Ig constant region or a portion thereof andthe additional Ig constant region or a portion thereof are associated bya chemical association or a physical association. In one embodiment, thechemical association, i.e., (:), is at least one non-peptide bond. Incertain embodiments, the chemical association, i.e., (:), is a covalentbond. In other embodiments, the chemical association, i.e., (:), is anon-covalent interaction, e.g., an ionic interaction, a hydrophobicinteraction, a hydrophilic interaction, a Van der Waals interaction, ora hydrogen bond. In other embodiments, (:) is a non-peptide covalentbond. In still other embodiments, (:) is a peptide bond. In yet otherembodiments, (:) represents a physical association between twosequences, wherein a portion of a first sequence is in close proximityto a second sequence such that the first sequence shields or blocks aportion of the second sequence from interacting with another moiety. Insome embodiments, the association between the Ig constant region or aportion thereof and the additional Ig constant region or a portionthereof can be a covalent bond, e.g., a disulfide bond, which preventsreplacement the VWF fragment or the polypeptide containing the VWFfragment with endogenous VWF. Therefore, preventing interaction betweenthe FVIII protein and endogenous VWF reduces or eliminates thishalf-life limiting factor for the FVIII protein, and thus the half-lifeof the FVIII protein is extended compared to a FVIII protein without theVWF protein or wild-type FVIII.

In other aspects, a chimeric protein comprises a formula comprising:

FVIII(X1)-L1-F1:V-L2-X2-L3-F2;  (1)

FVIII(X1)-L1-F1:F2-L3-X2-L2-V;  (2)

F1-L1-FVIII(X1):V-L2-X2-L3-F2;  (3)

F1-L1-FVIII(X1):F2-L3-X2-L2-V;  (4)

FVIII(X1)-L1-F1-L4-V-L2-X2-L3-F2;  (5)

FVIII(X1)-L1-F1-L4-F2-L3-X2-L2-V;  (6)

F1-L1-FVIII(X1)-L4-V-L2-X2-L3-F2, or  (7)

F1-L1-FVIII(X1)-L4-F2-L3-X2-L2-V,  (8)

wherein FVIII(X1) comprises a FVIII protein and one or more XTENsequences, wherein the one or more XTEN sequence are linked to theN-terminus or C-terminus of the FVIII protein or inserted immediatelydownstream of one or more amino acids (e.g., one or more XTEN insertionsites disclosed herein) in the FVIII protein;each of L1, L2, or L3 comprises an optional linker, e.g., a cleavablelinker;L4 is a linker, e.g., a processable linker;X2 comprises one or more optional XTEN sequences;F1 comprises an Ig constant region or a portion thereof;F2 comprises an optional additional Ig constant region or a portionthereof, andV comprises a VWF fragment;(-) is a peptide bond or one or more amino acids; and(:) comprises a chemical association or a physical association. In oneembodiment, (:) represents a chemical association, e.g., at least onenon-peptide bond. In another embodiment, the chemical association, i.e.,(:) is a covalent bond. In other embodiments, the chemical association,i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, ahydrophobic interaction, a hydrophilic interaction, a Van der Waalsinteraction, or a hydrogen bond. In other embodiments, (:) is anon-peptide covalent bond. In still other embodiments, (:) is a peptidebond. In yet other embodiments, (:) represents a physical associationbetween two sequences, wherein a portion of a first sequence is in closeproximity to a second sequence such that the first sequence shields orblocks a portion of the second sequence from interacting with anothermoiety, and further that this physical association is maintained withoutallowing the second sequence to interact with other moieties. Theorientation of the polypeptide formulas herein is listed from N-terminus(left) to C-terminus (right). For example, formula V-X-FVIII meansformula NH2-V-X-FVIII-COOH. In one embodiment, the formulas describedherein can comprise any additional sequences between the two moieties.For example, formula V-X-FVIII can further comprise any sequences at theN-terminus of V between V and X, between X and FVIII, or at theC-terminus of FVIII unless otherwise specified. In another embodiment,the hyphen (-) indicates a peptide bond.

In one aspect, the chimeric protein comprises two polypeptide chains,(A) a first chain comprising (i) a single chain FVIII protein (ii) anXTEN sequence, and (iii) a first Ig constant region or a portionthereof, e.g., a first Fc region or FcRn binding partner, wherein theXTEN sequence is linked to the FVIII protein at the N-terminus orC-terminus or inserted immediately downstream of one or more amino acidsof the FVIII protein (e.g., one or more XTEN insertion sites disclosedherein) and the first Ig constant region or a portion thereof is linkedto the XTEN sequence when the XTEN sequence is linked to the FVIIIprotein at the N-terminus or the C-terminus or the FVIII protein whenthe XTEN sequence is inserted within the FVIII protein, and (B) a secondchain comprising (iv) a VWF fragment comprising a D′ domain and a D3domain, (v) a linker, and (vi) a second Ig constant region or a portionthereof, e.g., a second Fc region or a second FcRn binding partner,wherein the VWF fragment is linked to the linker, e.g., a cleavablelinker, which is further linked to the second Ig constant region or aportion thereof, and wherein the first polypeptide chain and the secondpolypeptide chain are associated with each other, e.g., a covalent bond,e.g., a disulfide bond. In one embodiment, the linker is a cleavablelinker described elsewhere herein, e.g., a thrombin cleavable linker. Insome embodiments, the second chain comprises one or more XTEN sequencesbetween (iv) and (v) or (v) and (vi).

In other aspects, the chimeric protein comprises one polypeptide chaincomprising (i) a single chain FVIII protein (ii) an XTEN sequence, (iii)a first Ig constant region or a portion thereof, e.g., a first Fc regionor a first FcRn binding partner, (iv) a first linker, (v) a VWF fragmentcomprising a D′ domain and a D3 domain, (vi) a second linker, and (vii)a second Ig constant region or a portion thereof, e.g., a second Fcregion or a second FcRn binding partner, wherein (i) to (vii) are linkedin the order or in any orders. In one embodiment, the first linker is aprocessable linker, which can be intracellularly processed or cleavedafter expression and makes the single polypeptide chain into twopolypeptide chains. In another embodiment, the second linker is acleavable linker described herein, e.g., a thrombin cleavable linker.The XTEN sequence used herein can be linked to the FVIII protein by anoptional linker at the N-terminus or the C terminus of the FVIII proteinor inserted immediately downstream of one or more amino acids (e.g., oneor more XTEN insertion sites) in the FVIII protein.

In certain aspects, a chimeric protein comprises three polypeptidechains, (A) a first polypeptide chain comprising (i) a heavy chain of aFVIII protein and (ii) an XTEN sequence, which are linked to each otherand (B) a second polypeptide chain comprising (iii) a light chain of theFVIII protein and (iv) a first Ig constant region or a portion thereof,e.g., a first Fc region or a first FcRn binding partner, which arelinked to each other, and (C) a third polypeptide chain comprising (v) aVWF fragment comprising a D′ domain and a D3 domain, (vi) a linker, and(vii) a second Ig constant region or a portion thereof, e.g., a secondFc region or a second FcRn binding partner, wherein the second chain isassociated with the first chain and the third chain. In one embodiment,the association between the first chain and the second chain is achemical association or a physical association. For example, theassociation between the first chain and the second chain can be a metalbond. In another embodiment, the association between the second chainand the third chain is also a chemical association or a physicalassociation, e.g., a covalent bond or a non-covalent bond. In certainembodiments, the association between the second chain and the thirdchain is through the two Ig constant regions or a portion thereof and isa disulfide bond. The bonding between the second chain and the thirdchain prevents or inhibits binding of the FVIII protein with endogenousVWF, thus preventing the FVIII protein being cleared by the VWFclearance pathway. In some embodiments, the linker is a processablelinker, which is intracellularly cleaved after expression in a hostcell. The XTEN sequence used herein is linked to the FVIII protein by anoptional linker at the N-terminus or C terminus of the FVIII protein orinserted immediately downstream of one or more amino acids (e.g., one ormore XTEN insertion sites) in the FVIII protein.

In certain embodiments, the VWF fragment is directly linked to the FVIIIprotein, which comprises one or more XTENs, by a peptide bond or alinker. As one way of linking the VWF fragment and the FVIII protein, inwhich one or more XTENs are inserted or linked, through a direct link(e.g. a peptide bond) or a linker, an enzymatic ligation (e.g., sortase)can be employed. For example, sortase refers to a group of prokaryoticenzymes that modify surface proteins by recognizing and cleaving acarboxyl-terminal sorting signal. For most substrates of sortaseenzymes, the recognition signal consists of the motif LPXTG(Leu-Pro-any-Thr-Gly (SEQ ID NO: 51), then a highly hydrophobictransmembrane sequence, then a cluster of basic residues such asarginine. Cleavage occurs between the Thr and Gly, with transientattachment through the Thr residue to the active site Cys residue of aligation partner, followed by transpeptidation that attaches the proteincovalently to the cell wall. In some embodiments, the ligation partnercontains Gly(n). In other embodiments, the chimeric protein furthercomprises a sortase recognition motif. In some embodiments, the VWFfragment is attached to FVIII comprising one or more XTENs insertedwithin or linked to using sortase mediated in vitro protein ligation.

In one embodiment, a VWF fragment linked to a sortase recognition motifby an optional linker can be fused to a FVIII protein linked to Gly(n)by a sortase, wherein n can be any integer and wherein one or more XTENsare inserted within or linked to the FVIII protein. A ligation constructcomprises the VWF fragment (N-terminal portion of the construct) and theFVIII protein, in which one or more XTENs are inserted or linked(C-terminal portion of the construct), wherein the sortase recognitionmotif is inserted in between. Another ligation construct comprises theVWF fragment (N-terminal portion of the construct, the linker, thesortase recognition motif, and the FVIII protein, in which one or moreXTENs are inserted or linked (C-terminal portion of the construct). Inanother embodiment, a FVIII protein linked to a sortase recognitionmotif by an optional linker can be fused to a VWF fragment linked toGly(n) by a sortase, wherein n is any integer. A resulting ligationconstruct comprises the FVIII protein (N-terminal portion of theconstruct), in which one or more XTENs are inserted or linked, and theVWF fragment (C-terminal portion of the construct), wherein the sortaserecognition motif is inserted in between. Another resulting ligationconstruct comprises the FVIII protein (N-terminal portion of theconstruct), in which one or more XTENs are inserted or linked, thelinker, the sortase recognition motif, and the VWF fragment (C-terminalportion of the construct). In other embodiments, a VWF fragment linkedto a sortase recognition motif by a first optional linker can be fusedto a heterologous moiety, e.g., an immunoglobulin constant region or aportion thereof, e.g., an Fc region, linked to a thrombin cleavage siteby a second optional linker. A resulting construct can comprise the VWFfragment (N-terminal portion), the first linker, the sortase recognitionmotif, the protease cleavage site, the second optional linker, and theheterologous moiety.

In some embodiments, the VWF fragment is associated with the FVIIIprotein. The association between the VWF fragment and the FVIII proteincan be a chemical association or a physical association. The chemicalassociation can be a non-covalent interaction, e.g., an ionicinteraction, a hydrophobic interaction, a hydrophilic interaction, a Vander Waals interaction, or a hydrogen bond. In yet other embodiments, theassociation between the FVIII protein and the VWF fragment is a physicalassociation between two sequences, e.g., due to an additionalassociation between the sequence having the FVIII protein and thesequence having the VWF fragment, wherein a portion of a first sequenceis in close proximity to a second sequence such that the first sequenceshields or blocks a portion of the second sequence from interacting withanother moiety.

As a result of preventing or inhibiting endogenous VWF interaction withthe FVIII protein by the VWF fragment, the chimeric protein describedherein have an extended half-life compared to wild-type FVIII or thecorresponding chimeric protein without the VWF fragment. In oneembodiment, the half-life of the FVIII protein is extended at leastabout 1.5 times, at least about 2 times, at least about 2.5 times, atleast about 3 times, at least about 4 times, at least about 5 times, atleast about 6 times, at least about 7 times, at least about 8 times, atleast about 9 times, at least about 10 times, at least about 11 times,or at least about 12 times longer than a FVIII protein without the VWFfragment. In another embodiment, the half-life of the FVIII protein isat least about 10 hours, at least about 11 hours, at least about 12hours, at least about 13 hours, at least about 14 hours, at least about15 hours, at least about 16 hours, at least about 17 hours, at leastabout 18 hours, at least about 19 hours, at least about 20 hours, atleast about 21 hours, at least about 22 hours, at least about 23 hours,at least about 24 hours, at least about 36 hours, at least about 48hours, at least about 60 hours, at least about 72 hours, at least about84 hours, at least about 96 hours, or at least about 108 hours. In aparticular embodiment, the half-life of the FVIII protein is extended atleast 10 hours, at least about 11 hours, at least about 12 hours, atleast about 13 hours, at least about 14 hours, at least about 15 hours,at least about 16 hours, at least about 17 hours, at least about 18hours, at least about 19 hours, at least about 20 hours, at least about21 hours, at least about 22 hours, at least about 23 hours, at leastabout 24 hours, at least about 25 hours, at least about 26 hours, or atleast about 27 hours in HemA mice.

A) Von Willebrand Factor (VWF) Fragments

VWF (also known as F8VWF) is a large multimeric glycoprotein present inblood plasma and produced constitutively in endothelium (in theWeibel-Palade bodies), megakaryocytes (α-granules of platelets), andsubendothelian connective tissue. The basic VWF monomer is a 2813 aminoacid protein. Every monomer contains a number of specific domains with aspecific function, the D′/D3 domain (which binds to Factor VIII), the A1domain (which binds to platelet GPIb-receptor, heparin, and/or possiblycollagen), the A3 domain (which binds to collagen), the C1 domain (inwhich the RGD domain binds to platelet integrin αIIbβ3 when this isactivated), and the “cysteine knot” domain at the C-terminal end of theprotein (which VWF shares with platelet-derived growth factor (PDGF),transforming growth factor-β (TGFβ) and β-human chorionic gonadotropin(βHCG).

The term “a VWF fragment” as used herein includes, but is not limitedto, functional VWF fragments comprising a D′ domain and a D3 domain,which are capable of inhibiting binding of endogenous VWF to FVIII. Inone embodiment, the VWF fragment binds to the FVIII protein. In anotherembodiment, the VWF fragment blocks the VWF binding site on the FVIIIprotein, thereby inhibiting interaction of the FVIII protein withendogenous VWF. The VWF fragments include derivatives, variants,mutants, or analogues that retain these activities of VWF.

The 2813 monomer amino acid sequence for human VWF is reported asAccession Number_NP_000543.2_ in Genbank. The nucleotide sequenceencoding the human VWF is reported as Accession Number _NM_000552.3_ inGenbank. The nucleotide sequence of human VWF is designated as SEQ IDNO: 1. SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1.Each domain of VWF is listed in Table 1.

TABLE 1 VWF Sequences VWF domains Amino acid Sequence VWF Signal Peptide1 MIPARFAGVL LALALILPGT LC (Amino acids 1 to 22 22 of SEQ ID NO: 2)VWF D1D2 region 23                        AEGTRGRS (Amino acids 23 toSTARCSLFGS DFVNTFDGSM 763 of SEQ ID NO: 2) 51YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG 101TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGEVARI    DGSGNFQVLL 151SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC 201ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC 251EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME 301YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC 351VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD 401NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG 451LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM 501DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG 551NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS 601PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL 651NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD 701CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD 751AVLSSPLSHR                    SKR 763 VWF D′ Domain 764                SLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM 801SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV 851CRDRKWNCTD         HVCDAT 866 VWF D3 Domain 867                   

901

951

1001

1051

1101

1151

1201

1240 VWF A1 Domain 1241  GGLVVPPTDA 1251PVSPTTLYVE DISEPPLHDF YCSRLLDLVF LLDGSSRLSE AEFEVLKAFV 1301VDMMERLRIS QKWVRVAVVE YHDGSHAYIG LKDRKRPSEL RRIASQVKYA 1351GSQVASTSEV LKYTLFQIFS KIDRPEASRI ALLLMASQEP QRMSRNFVRY 1401VQGLKKKKVI VIPVGIGPHA NLKQIRLIEK QAPENKAFVL SSVDELEQQR 1451DEIVSYLCDL APEAPPPTLP PDMAQVTVG 1479 1480                        PGLLGVSTLGP KRNSMVLDVA 1501 FVLEGSDKIG EADFNRSKEF MEEVIQRMDVGQDSIHVTVL QYSYMVTVEY 1551 PFSEAQSKGD ILQRVREIRY QGGNRTNTGLALRYLSDHSF LVSQGDREQA 1600 1601 PNLVYMVTGN PASDEIKRLP GDIQVVPIGVGPNANVQELE RIGWPNAPIL 1651 IQDFETLPRE APDLVLQRCC SGEGLQIPTLSPAPDCSQPL DVILLLDGSS 1701 SFPASYFDEM KSFAKAFISK ANIGPRLTQVSVLQYGSITT IDVPWNVVPE 1751 KAHLLSLVDV MQREGGPSQI GDALGFAVRYLTSEMHGARP GASKAVVILV 1801 TDVSVDSVDA AADAARSNRV TVFPIGIGDRYDAAQLRILA GPAGDSNVVK 1851 LQRIEDLPTM VTLGNSFLHK LCSGFVRICMDEDGNEKRPG DVWTLPDQCH 1901 TVTCQPDGQT LLKSHRVNCD RGLRPSCPNSQSPVKVEETC GCRWTCPCVC 1951 TGSSTRHIVT FDGQNFKLTG SCSYVLFQNKEQDLEVILHN GACSPGARQG 2001 CMKSIEVKHS ALSVEXHSDM EVTVNGRLVSVPYVGGNMEV NVYGAIMHEV 2051 RFNHLGHIFT FTPQNNEFQL QLSPKTFASKTYGLCGICDE NGANDFMLRD 2101 GTVTTDWKTL VQEWTVQRPG QTCQPILEEQCLVPDSSHCQ VLLLPLFAEC 2151 HKVLAPATFY AICQQDSCHQ EQVCEVIASYAHLCRTNGVC VDWRTPDFCA 2201 MSCPPSLVYN HCEHGCPRHC DGNVSSCGDHPSEGCFCPPD KVMLEGSCVP 2251 EEACTQCIGE DGVQHQFLEA WVPDHQPCQICTCLSGRKVN CTTQPCPTAK 2301 APTCGLCEVA RLRQNADQCC PEYECVCDPVSCDLPPVPHC ERGLQPTLTN 2351 PGECRPNFTC ACRKEECKRV SPPSCPPHRLPTLRKTQCCD EYECACNCVN 2401 STVSCPLGYL ASTATNDCGC TTTTCLPDKVCVHRSTIYPV GQFWEEGCDV 2451 CTCTDMEDAV MGLRVAQCSQ KPCEDSCRSGFTYVLHEGEC CGRCLPSACE 2501 VVTGSPRGDS QSSWKSVGSQ WASPENPCLINECVRVKEEV FIQQRNVSCP 2551 QLEVPVCPSG FQLSCKTSAC CPSCRCERMEACMLNGTVIG PGKTVMIDVC 2601 TTCRCMVQVG VISGFKLECR KTTCNPCPLGYKEENNTGEC CGRCLPTACT 2651 IQLRGGQIMT LKRDETLQDG CDTHFCKVNERGEYFWEKRV TGCPPFDEHK 2701 CLAEGGKIMK IPGTCCDTCE EPECNDITARLQYVKVGSCK SEVEVDIHYC 2751 QGKCASKAMY SIDINDVQDQ CSCCSPTRTEPMQVALHCTN GSVVYHEVLN 2801 AMECKCSPRK CSKNucleotide Sequence (SEQ ID NO: 1) Full-length VWF 1       ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG CCCTCATTTT 51GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC 101GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG 151TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA 201ACGCTCCTTC TCGATTATTG GGGACTTCCA GAATGGCAAG AGAGTGAGCC 251TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT 301ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG 351GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCTGTCC GGTGAGGCCT 401ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG 451TCAGACAGAT ACTTCAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT 501CTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTG ACCTCGGACC 551CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT 601GAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATCTCCT CTGGGGAAAT 651GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT 701TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT 751GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC 801CCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG 851GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG 901TATAGGCAGT GTGTGTCCCC TTGCGCCAGG ACCTGCCAGA GCCTGCACAT 951CAATGAAATG TGTCAGGAGC GATGCGTGGA TGGCTGCAGC TGCCCTGAGG 1001GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCTGC 1051GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG 1101CAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAAT 1151GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC 1201AACAGATACT TCACCTTCAG TGGGATCTGC CAGTACCTGC TGGCCCGGGA 1251TTGCCAGGAC CACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG 1301ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC 1351CTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGA 1401TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC 1451ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG 1501GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC 1551CGGGAAGACC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACG 1601ACTTCCTTAC CCCCTCTGGG CTGGCRGAGC CCCGGGTGGA GGACTTCGGG 1651AACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGA AGCAGCACAG 1701CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT 1751GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801CCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA 1851CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG 1901CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG 1951AACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCT 2001GACCTGCCGC TCTCTCTCTT ACCCGGATGA GGAATGCAAT GAGGCCTGCC 2051TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151GCCAGAAGAC ATCTTCTCAG ACCATCACAC CATGTGCTAC TGTGAGGATG 2201GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC 2251GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG 2301TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG 2351AAGGGCTCGA GTGTACCAAA ACGTGCCAGA ACTATGACCT GGAGTGCATG 2401AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA 2451TGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA 2501AGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTC 2551TGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTGTG ATGCCACGTG 2601CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACC 2651TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT 2701AACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGGAT GCAGCCACCC 2751CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA 2801TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG 2851ACTCACTTTG AGGTGGTGGA GTCTGGCCGG TACATCATTC TGCTGCTGGG 2901CAAAGCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATC TCCGTGGTCC 2951TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT 3001GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA 3051CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA 3101CCAGAAAAGT GCCTCTGGAC TCATCCCCTG CCACCTGCCA TAACAACATC 3151ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT 3201CTTCCAGGAC TGCAACAAGC TGGTGGACCC CGAGCCATAT CTGGATGTCT 3251GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCTGCTTC 3301TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGT 3351GGTGACCTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGA 3401ATCTCCGGGA GAACGGGTAT GAGTGTGAGT GGCGCTATAA CAGCTGTGCA 3451CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT 3501GCAGTGTGTG GAGGGCTGCC ATGCCCACTG CCCTCCAGGG AAAATCCTGG 3551ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAG 3601GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG 3651TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT 3701GTGAAGCCTG CCAGGAGCCG GGAGGCCTGG TGGTGCCTCC CACAGATGCC 3751CCGGTGAGCC CCACCACTCT GTATGTGGAG GACATCTCGG AACCGCCGTT 3801GCACGATTTC TACTGCAGCA GGCTACTGGA CCTGGTCTTC CTGCTGGATG 3851GCTCCTCCAG GCTGTCCGAG GCTGAGTTTG AAGTGCTGAA GGCCTTTGTG 3901GTGGACATGA TGGAGCGGCT GCGCATCTCC CAGAAGTGGG TCCGCGTGGC 3951CGTGGTGGAG TACCACGACG GCTCCCACGC CTACATCGGG CTCAAGGACC 4001GGAAGCGACC GTCAGAGCTG CGGCGCATTG CCAGCCAGGT GAAGTATGCG 4051GGCAGCCAGG TGGCCTCCAC CAGCGAGGTC TTGAAATACA CACTGTTCCA 4101AATCTTCAGC AAGATCGACC GCCCTGAAGC CTCCCGCATC GCCCTGCTCC 4151TGATGGCCAG CCAGGAGCCC CAACGGATGT CCCGGAACTT TGTCCGCTAC 4201GTCCAGGGCC TGAAGAAGAA GAAGGTCATT GTGATCCCGG TGGGCATTGG 4251GCCCCATGCC AACCTCAAGC AGATCCGCCT CATCGAGAAG CAGGCCCCTG 4301AGAACAAGGC CTTCGTGCTG AGCAGTGTGG ATGAGCTGGA GCAGCAAAGG 4351GACGAGATCG TTAGCTACCT CTGTGACCTT GCCCCTGAAG CCCCTCCTCC 4401TACTCTGCCC CCCGACATGG CACAAGTCAC TGTGGGCCCG GGGCTCTTGG 4451GGGTTTCGAC CCTGGGGCCC AAGAGGAACT CCATGGTTCT GGATGTGGCG 4501TTCGTCCTGG AAGGATCGGA CAAAATTGGT GAAGCCGACT TCAACAGGAG 4551CAAGGAGTTC ATGGAGGAGG TGATTCAGCG GATGGATGTG GGCCAGGACA 4601GCATCCACGT CACGGTGCTG CAGTACTCCT ACATGGTGAC CGTGGAGTAC 4651CCCTTCAGCG AGGCACAGTC CAAAGGGGAC ATCCTGCAGC GGGTGCGAGA 4701GATCCGCTAC CAGGGCGGCA ACAGGACCAA CACTGGGCTG GCCCTGCGGT 4751ACCTCTCTGA CCACAGCTTC TTGGTCAGCC AGGGTGACCG GGAGCAGGCG 4801CCCAACCTGG TCTACATGGT CACCGGAAAT CCTGCCTCTG ATGAGATCAA 4851GAGGCTGCCT GGAGACATCC AGGTGGTGCC CATTGGAGTG GGCCCTAATG 4901CCAACGTGCA GGAGCTGGAG AGGATTGGCT GGCCCAATGC CCCTATCCTC 4951ATCCAGGACT TTGAGACGCT CCCCCGAGAG GCTCCTGACC TGGTGCTGCA 5001GAGGTGCTGC TCCGGAGAGG GGCTGCAGAT CCCCACCCTC TCCCCTGCAC 5051CTGACTGCAG CCAGCCCCTG GACGTGATCC TTCTCCTGGA TGGCTCCTCC 5101AGTTTCCCAG CTTCTTATTT TGATGAAATG AAGAGTTTCG CCAAGGCTTT 5151CATTTCAAAA GCCAATATAG GGCCTCGTCT CACTCAGGTG TCAGTGCTGC 5201AGTATGGAAG CATCACCACC ATTGACGTGC CATGGAACGT GGTCCCGGAG 5251AAAGCCCATT TGCTGAGCCT TGTGGACGTC ATGCAGCGGG AGGGAGGCCC 5301CAGCCAAATC GGGGATGCCT TGGGCTTTGC TGTGCGATAC TTGACTTCAG 5351AAATGCATGG TGCCAGGCCG GGAGCCTCAA AGGCGGTGGT CATCCTGGTC 5401ACGGACGTCT CTGTGGATTC AGTGGATGCA GCAGCTGATG CCGCCAGGTC 5451CAACAGAGTG ACAGTGTTCC CTATTGGAAT TGGAGATCGC TACGATGCAG 5501CCCAGCTACG GATCTTGGCA GGCCCAGCAG GCGACTCCAA CGTGGTGAAG 5551CTCCAGCGAA TCGAAGACCT CCCTACCATG GTCACCTTGG GCAATTCCTT 5601CCTCCACAAA CTGTGCTCTG GATTTGTTAG GATTTGCATG GATGAGGATG 5651GGAATGAGAA GAGGCCCGGG GACGTCTGGA CCTTGCCAGA CCAGTGCCAC 5701ACCGTGACTT GCCAGCCAGA TGGCCAGACC TTGCTGAAGA GTCATCGGGT 5751CAACTGTGAC CGGGGGCTGA GGCCTTCGTG CCCTAACAGC CAGTCCCCTG 5801TTAAAGTGGA AGAGACCTGT GGCTGCCGCT GGACCTGCCC CTGYGTGTGC 5851ACAGGCAGCT CCACTCGGCA CATCGTGACC TTTGATGGGC AGAATTTCAA 5901GCTGACTGGC AGCTGTTCTT ATGTCCTATT TCAAAACAAG GAGCAGGACC 5951TGGAGGTGAT TCTCCATAAT GGTGCCTGCA GCCCTGGAGC AAGGCAGGGC 6001TGCATGAAAT CCATCGAGGT GAAGCACAGT GCCCTCTCCG TCGAGSTGCA 6051CAGTGACATG GAGGTGACGG TGAATGGGAG ACTGGTCTCT GTTCCTTACG 6101TGGGTGGGAA CATGGAAGTC AACGTTTATG GTGCCATCAT GCATGAGGTC 6151AGATTCAATC ACCTTGGTCA CATCTTCACA TTCACTCCAC AAAACAATGA 6201GTTCCAACTG CAGCTCAGCC CCAAGACTTT TGCTTCAAAG ACGTATGGTC 6251TGTGTGGGAT CTGTGATGAG AACGGAGCCA ATGACTTCAT GCTGAGGGAT 6301GGCACAGTCA CCACAGACTG GAAAACACTT GTTCAGGAAT GGACTGTGCA 6351GCGGCCAGGG CAGACGTGCC AGCCCATCCT GGAGGAGCAG TGTCTTGTCC 6401CCGACAGCTC CCACTGCCAG GTCCTCCTCT TACCACTGTT TGCTGAATGC 6451CACAAGGTCC TGGCTCCAGC CACATTCTAT GCCATCTGCC AGCAGGACAG 6501TTGCCACCAG GAGCAAGTGT GTGAGGTGAT CGCCTCTTAT GCCCACCTCT 6551GTCGGACCAA CGGGGTCTGC GTTGACTGGA GGACACCTGA TTTCTGTGCT 6601ATGTCATGCC CACCATCTCT GGTCTACAAC CACTGTGAGC ATGGCTGTCC 6651CCGGCACTGT GATGGCAACG TGAGCTCCTG TGGGGACCAT CCCTCCGAAG 6701GCTGTTTCTG CCCTCCAGAT AAAGTCATGT TGGAAGGCAG CTGTGTCCCT 6751GAAGAGGCCT GCACTCAGTG CATTGGTGAG GATGGAGTCC AGCACCAGTT 6801CCTGGAAGCC TGGGTCCCGG ACCACCAGCC CTGTCAGATC TGCACATGCC 6851TCAGCGGGCG GAAGGTCAAC TGCACAACGC AGCCCTGCCC CACGGCCAAA 6901GCTCCCACGT GTGGCCTGTG TGAAGTAGCC CGCCTCCGCC AGAATGCAGA 6951CCAGTGCTGC CCCGAGTATG AGTGTGTGTG TGACCCAGTG AGCTGTGACC 7001TGCCCCCAGT GCCTCACTGT GAACGTGGCC TCCAGCCCAC ACTGACCAAC 7051CCTGGCGAGT GCAGACCCAA CTTCACCTGC GCCTGCAGGA AGGAGGAGTG 7101CAAAAGAGTG TCCCCACCCT CCTGCCCCCC GCACCGTTTG CCCACCCTTC 7151GGAAGACCCA GTGCTGTGAT GAGTATGAGT GTGCCTGCAA CTGTGTCAAC 7201TCCACAGTGA GCTGTCCCCT TGGGTACTTG GCCTCAACCG CCACCAATGA 7251CTGTGGCTGT ACCACAACCA CCTGCCTTCC CGACAAGGTG TGTGTCCACC 7301GAAGCACCAT CTACCCTGTG GGCCAGTTCT GGGAGGAGGG CTGCGATGTG 7351TGCACCTGCA CCGACATGGA GGATGCCGTG ATGGGCCTCC GCGTGGCCCA 7401GTGCTCCCAG AAGCCCTGTG AGGACAGCTG TCGGTCGGGC TTCACTTACG 7451TTCTGCATGA AGGCGAGTGC TGTGGAAGGT GCCTGCCATC TGCCTGTGAG 7501GTGGTGACTG GCTCACCGCG GGGGGACTCC CAGTCTTCCT GGAAGAGTGT 7551CGGCTCCCAG TGGGCCTCCC CGGAGAACCC CTGCCTCATC AATGAGTGTG 7601TCCGAGTGAA GGAGGAGGTC TTTATACAAC AAAGGAACGT CTCCTGCCCC 7651CAGCTGGAGG TCCCTGTCTG CCCCTCGGGC TTTCAGCTGA GCTGTAAGAC 7701CTCAGCGTGC TGCCCAAGCT GTCGCTGTGA GCGCATGGAG GCCTGCATGC 7751TCAATGGCAC TGTCATTGGG CCCGGGAAGA CTGTGATGAT CGATGTGTGC 7801ACGACCTGCC GCTGCATGGT GCAGGTGGGG GTCATCTCTG GATTCAAGCT 7851GGAGTGCAGG AAGACCACCT GCAACCCCTG CCCCCTGGGT TACAAGGAAG 7901AAAATAACAC AGGTGAATGT TGTGGGAGAT GTTTGCCTAC GGCTTGCACC 7951ATTCAGCTAA GAGGAGGACA GATCATGACA CTGAAGCGTG ATGAGACGCT 8001CCAGGATGGC TGTGATACTC ACTTCTGCAA GGTCAATGAG AGAGGAGAGT 8051ACTTCTGGGA GAAGAGGGTC ACAGGCTGCC CACCCTTTGA TGAACACAAG 8101TGTCTTGCTG AGGGAGGTAA AATTATGAAA ATTCCAGGCA CCTGCTGTGA 8151CACATGTGAG GAGCCTGAGT GCAACGACAT CACTGCCAGG CTGCAGTATG 8201TCAAGGTGGG AAGCTGTAAG TCTGAAGTAG AGGTGGATAT CCACTACTGC 8251CAGGGCAAAT GTGCCAGCAA AGCCATGTAC TCCATTGACA TCAACGATGT 8301GCAGGACCAG TGCTCCTGCT GCTCTCCGAC ACGGACGGAG CCCATGCAGG 8351TGGCCCTGCA CTGCACCAAT GGCTCTGTTG TGTACCATGA GGTTCTCAAT 8401GCCATGGAGT GCAAATGCTC CCCCAGGAAG TGCAGCAAGT GA

The VWF fragment as used herein can be a VWF fragment comprising a D′domain and a D3 domain of VWF, wherein the VWF fragment binds to FactorVIII (FVIII) and inhibits binding of endogenous VWF (full-length VWF) toFVIII. The VWF fragment comprising the D′ domain and the D3 domain canfurther comprise a VWF domain selected from the group consisting of anA1 domain, an A2 domain, an A3 domain, a D1 domain, a D2 domain, a D4domain, a B1 domain, a B2 domain, a B3 domain, a C1 domain, a C2 domain,a CK domain, one or more fragments thereof, and any combinationsthereof. In one embodiment, a VWF fragment comprises, consistsessentially of, or consists of: (1) the D′ and D3 domains of VWF orfragments thereof; (2) the D1, D′, and D3 domains of VWF or fragmentsthereof; (3) the D2, D′, and D3 domains of VWF or fragments thereof, (4)the D1, D2, D′, and D3 domains of VWF or fragments thereof; or (5) theD1, D2, D′, D3, and A1 domains of VWF or fragments thereof.

The VWF fragment described herein does not contain a site binding to aVWF clearance receptor. In another embodiment, the VWF fragmentdescribed herein is not amino acids 764 to 1274 of SEQ ID NO: 2. The VWFfragment of the present invention can comprise any other sequenceslinked to or fused to the VWF fragment. For example, a VWF fragmentdescribed herein can further comprise a signal peptide.

In one embodiment, the VWF fragment binds to or is associated with aFVIII protein. By binding to or associating with a FVIII protein, a VWFfragment of the invention protects FVIII from protease cleavage andFVIII activation, stabilizes the heavy chain and light chain of FVIII,and prevents clearance of FVIII by scavenger receptors. In anotherembodiment, the VWF fragment binds to or associates with a FVIII proteinand blocks or prevents binding of the FVIII protein to phospholipid andactivated Protein C. By preventing or inhibiting binding of the FVIIIprotein with endogenous, full-length VWF, the VWF fragment of theinvention reduces the clearance of FVIII by VWF clearance receptors andthus extends half-life of the FVIII protein. In one embodiment, thehalf-life extension of a FVIII protein is thus due to the binding of orassociating with the VWF fragment lacking a VWF clearance receptorbinding site to the FVIII protein and shielding or protecting of theFVIII protein by the VWF fragment from endogenous VWF which contains theVWF clearance receptor binding site. The FVIII protein bound to orprotected by the VWF fragment can also allow recycling of a FVIIIprotein. By eliminating the VWF clearance pathway receptor binding sitescontained in the full length VWF molecule, the FVIII/VWF heterodimers ofthe invention are shielded from the VWF clearance pathway, furtherextending FVIII half-life.

In one embodiment, a VWF fragment of the present invention comprises theD′ domain and the D3 domain of VWF, wherein the D′ domain is at least60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical toamino acids 764 to 866 of SEQ ID NO: 2, wherein the VWF fragmentprevents binding of endogenous VWF to FVIII. In another embodiment, aVWF fragment comprises the D′ domain and the D3 domain of VWF, whereinthe D3 domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 2,wherein the VWF fragment prevents binding of endogenous VWF to FVIII. Insome embodiments, a VWF fragment described herein comprises, consistsessentially of, or consists of the D′ domain and D3 domain of VWF, whichare at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 764 to 1240 of SEQ ID NO: 2, wherein the VWFfragment prevents binding of endogenous VWF to FVIII. In otherembodiments, a VWF fragment comprises, consists essentially of, orconsists of the D1, D2, D′, and D3 domains at least 60%, 70%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 23 to1240 of SEQ ID NO: 2, wherein the VWF fragment prevents binding ofendogenous VWF to FVIII. In still other embodiments, the VWF fragmentfurther comprises a signal peptide operably linked thereto.

In some embodiments, a VWF fragment of the invention consistsessentially of or consists of (1) the D′D3 domain, the D1D′D3 domain,D2D′D3 domain, or D1D2D′D3 domain and (2) an additional VWF sequence upto about 10 amino acids (e.g., any sequences from amino acids 764 to1240 of SEQ ID NO: 2 to amino acids 764 to 1250 of SEQ ID NO: 2), up toabout 15 amino acids (e.g., any sequences from amino acids 764 to 1240of SEQ ID NO: 2 to amino acids 764 to 1255 of SEQ ID NO: 2), up to about20 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQID NO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2), up to about 25amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ IDNO: 2 to amino acids 764 to 1265 of SEQ ID NO: 2), or up to about 30amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ IDNO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2). In a particularembodiment, the VWF fragment comprising or consisting essentially of theD′ domain and the D3 domain is neither amino acids 764 to 1274 of SEQ IDNO: 2 nor the full-length mature VWF. In some embodiments, the D1D2domain is expressed in trans with the D′D3 domain. In some embodiments,the D1D2 domain is expressed in cis with the D′D3 domain.

In other embodiments, the VWF fragment comprising the D′D3 domainslinked to the D1D2 domains further comprises an intracellular cleavagesite, e.g., (a cleavage site by PACE (furin) or PC5), allowing cleavageof the D1D2 domains from the D′D3 domains upon expression. Non-limitingexamples of the intracellular cleavage site are disclosed elsewhereherein.

In yet other embodiments, a VWF fragment comprises the D′ domain and theD3 domain, but does not comprise an amino acid sequence selected fromthe group consisting of (1) amino acids 1241 to 2813 of SEQ ID NO: 2,(2) amino acids 1270 to amino acids 2813 of SEQ ID NO: 2, (3) aminoacids 1271 to amino acids 2813 of SEQ ID NO: 2, (4) amino acids 1272 toamino acids 2813 of SEQ ID NO: 2, (5) amino acids 1273 to amino acids2813 of SEQ ID NO: 2, (6) amino acids 1274 to amino acids 2813 of SEQ IDNO: 2, and any combinations thereof.

In still other embodiments, a VWF fragment of the present inventioncomprises, consists essentially of, or consists of an amino acidsequence corresponding to the D′ domain, D3 domain, and A1 domain,wherein the amino acid sequence is at least 60%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acid 764 to1479 of SEQ ID NO: 2, wherein the VWF fragment prevents binding ofendogenous VWF to FVIII. In a particular embodiment, the VWF fragment isnot amino acids 764 to 1274 of SEQ ID NO: 2.

In some embodiments, a VWF fragment of the invention comprises the D′domain and the D3 domain, but does not comprise at least one VWF domainselected from the group consisting of (1) an A1 domain, (2) an A2domain, (3) an A3 domain, (4) a D4 domain, (5) a B1 domain, (6) a B2domain, (7) a B3 domain, (8) a C1 domain, (9) a C2 domain, (10) a CKdomain, (11) a CK domain and C2 domain, (12) a CK domain, a C2 domain,and a C1 domain, (13) a CK domain, a C2 domain, a C1 domain, a B3domain, (14) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2domain, (15) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2domain, and a B1 domain, (16) a CK domain, a C2 domain, a C1 domain, aB3 domain, a B2 domain, a B1 domain, and a D4 domain, (17) a CK domain,a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4domain, and an A3 domain, (18) a CK domain, a C2 domain, a C1 domain, aB3 domain, a B2 domain, a B1 domain, a D4 domain, an A3 domain, and anA2 domain, (19) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2domain, a B1 domain, a D4 domain, an A3 domain, an A2 domain, and an A1domain, and (20) any combinations thereof.

In yet other embodiments, the VWF fragment comprises the D′D3 domainsand one or more domains or modules. Examples of such domains or modulesinclude, but are not limited to, the domains and modules disclosed inZhour et al., Blood published online Apr. 6, 2012: DOI10.1182/blood-2012-01-405134. For example, the VWF fragment can comprisethe D′D3 domain and one or more domains or modules selected from thegroup consisting of A1 domain, A2 domain, A3 domain, D4N module, VWD4module, C8-4 module, TIL-4 module, C1 module, C2 module, C3 module, C4module, C5 module, C5 module, C6 module, and any combinations thereof.

In still other embodiments, the VWF fragment is linked to a heterologousmoiety, wherein the heterologous moiety is linked to the N-terminus orthe C-terminus of the VWF fragment or inserted immediately downstream ofone or more amino acids (e.g., one or more XTEN insertion sites) in theFVIII protein in the VWF fragment. For example, the insertion sites forthe heterologous moiety in the VWF fragment can be in the D′ domain, theD3 domain, or both. The heterologous moiety can be a half-life extender.

In certain embodiments, a VWF fragment of the invention forms amultimer, e.g., dimer, trimer, tetramer, pentamer, hexamer, heptamer, orthe higher order multimers. In other embodiments, the VWF fragment is amonomer having only one VWF fragment. In some embodiments, the VWFfragment of the present invention can have one or more amino acidsubstitutions, deletions, additions, or modifications. In oneembodiment, the VWF fragment can include amino acid substitutions,deletions, additions, or modifications such that the VWF fragment is notcapable of forming a disulfide bond or forming a dimer or a multimer. Inanother embodiment, the amino acid substitution is within the D′ domainand the D3 domain. In a particular embodiment, a VWF fragment of theinvention contains at least one amino acid substitution at a residuecorresponding to residue 1099, residue 1142, or both residues 1099 and1142 of SEQ ID NO: 2. The at least one amino acid substitution can beany amino acids that are not occurring naturally in the wild type VWF.For example, the amino acid substitution can be any amino acids otherthan cysteine, e.g., Isoleucine, Alanine, Leucine, Asparagine, Lysine,Aspartic acid, Methionine, Phenylalanine, Glutamic acid, Threonine,Glutamine, Tryptophan, Glycine, Valine, Proline, Serine, Tyrosine,Arginine, or Histidine. In another example, the amino acid substitutionhas one or more amino acids that prevent or inhibit the VWF fragmentsfrom forming multimers.

In certain embodiments, the VWF fragment useful herein can be furthermodified to improve its interaction with FVIII, e.g., to improve bindingaffinity to FVIII. As a non-limiting example, the VWF fragment comprisesa serine residue at the residue corresponding to amino acid 764 of SEQID NO: 2 and a lysine residue at the residue corresponding to amino acid773 of SEQ ID NO: 2. Residues 764 and/or 773 can contribute to thebinding affinity of the VWF fragments to FVIII. In other embodiments,the VWF fragments useful for the invention can have other modifications,e.g., the protein can be pegylated, glycosylated, hesylated, orpolysialylated.

B) XTEN Sequences

As used here “XTEN sequence” refers to extended length polypeptides withnon-naturally occurring, substantially non-repetitive sequences that arecomposed mainly of small hydrophilic amino acids, with the sequencehaving a low degree or no secondary or tertiary structure underphysiologic conditions. As a chimeric protein partner, XTENs can serveas a carrier, conferring certain desirable pharmacokinetic,physicochemical and pharmaceutical properties when linked to a VWFfragment or a FVIII sequence of the invention to create a chimericprotein. Such desirable properties include but are not limited toenhanced pharmacokinetic parameters and solubility characteristics. Asused herein, “XTEN” specifically excludes antibodies or antibodyfragments such as single-chain antibodies or Fc fragments of a lightchain or a heavy chain.

In some embodiments, the XTEN sequence of the invention is a peptide ora polypeptide having greater than about 20, 30, 40, 50, 60, 70, 80, 90,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acidresidues. In certain embodiments, XTEN is a peptide or a polypeptidehaving greater than about 20 to about 3000 amino acid residues, greaterthan 30 to about 2500 residues, greater than 40 to about 2000 residues,greater than 50 to about 1500 residues, greater than 60 to about 1000residues, greater than 70 to about 900 residues, greater than 80 toabout 800 residues, greater than 90 to about 700 residues, greater than100 to about 600 residues, greater than 110 to about 500 residues, orgreater than 120 to about 400 residues.

The XTEN sequence of the invention can comprise one or more sequencemotif of 9 to 14 amino acid residues or an amino acid sequence at least80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe sequence motif, wherein the motif comprises, consists essentiallyof, or consists of 4 to 6 types of amino acids selected from the groupconsisting of glycine (G), alanine (A), serine (S), threonine (T),glutamate (E) and proline (P). See US 2010-0239554 A1.

In some embodiments, the XTEN comprises non-overlapping sequence motifsin which about 80%, or at least about 85%, or at least about 90%, orabout 91%, or about 92%, or about 93%, or about 94%, or about 95%, orabout 96%, or about 97%, or about 98%, or about 99% or about 100% of thesequence consists of multiple units of non-overlapping sequencesselected from a single motif family selected from Table 2A, resulting ina family sequence. As used herein, “family” means that the XTEN hasmotifs selected only from a single motif category from Table 2A; i.e.,AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and that any other amino acidsin the XTEN not from a family motif are selected to achieve a neededproperty, such as to permit incorporation of a restriction site by theencoding nucleotides, incorporation of a cleavage sequence, or toachieve a better linkage to FVIII or VWF. In some embodiments of XTENfamilies, an XTEN sequence comprises multiple units of non-overlappingsequence motifs of the AD motif family, or of the AE motif family, or ofthe AF motif family, or of the AG motif family, or of the AM motiffamily, or of the AQ motif family, or of the BC family, or of the BDfamily, with the resulting XTEN exhibiting the range of homologydescribed above. In other embodiments, the XTEN comprises multiple unitsof motif sequences from two or more of the motif families of Table 2A.These sequences can be selected to achieve desired physical/chemicalcharacteristics, including such properties as net charge,hydrophilicity, lack of secondary structure, or lack of repetitivenessthat are conferred by the amino acid composition of the motifs,described more fully below. In the embodiments hereinabove described inthis paragraph, the motifs incorporated into the XTEN can be selectedand assembled using the methods described herein to achieve an XTEN ofabout 36 to about 3000 amino acid residues.

TABLE 2A XTEN Sequence Motifs of 12 Amino Acids and  Motif FamiliesMotif Family* MOTIF SEQUENCE AD GESPGGSSGSES AD GSEGSSGPGESS ADGSSESGSSEGGP AD GSGGEPSESGSS AE, AM GSPAGSPTSTEE AE, AM, AQ GSEPATSGSETPAE, AM, AQ GTSESATPESGP AE, AM, AQ GTSTEPSEGSAP AF, AM GSTSESPSGTAPAF, AM GTSTPESGSASP AF, AM GTSPSGESSTAP AF, AM GSTSSTAESPGP AG, AMGTPGSGTASSSP AG, AM GSSTPSGATGSP AG, AM GSSPSASTGTGP AG, AM GASPGTSSTGSPAQ GEPAGSPTSTSE AQ GTGEPSSTPASE AQ GSGPSTESAPTE AQ GSETPSGPSETA AQGPSETSTSEPGA AQ GSPSEPTEGTSA BC GSGASEPTSTEP BC GSEPATSGTEPS BCGTSEPSTSEPGA BC GTSTEPSEPGSA BD GSTAGSETSTEA BD GSETATSGSETA BDGTSESATSESGA BD GTSTEASEGSAS Denotes individual motif sequences that,when used together in various permutations, results in a ″familysequence″

XTEN can have varying lengths for insertion into or linkage to FVIII orVWF. In one embodiment, the length of the XTEN sequence(s) is chosenbased on the property or function to be achieved in the fusion protein.Depending on the intended property or function, XTEN can be short orintermediate length sequence or longer sequence that can serve ascarriers. In certain embodiments, the XTEN include short segments ofabout 6 to about 99 amino acid residues, intermediate lengths of about100 to about 399 amino acid residues, and longer lengths of about 400 toabout 1000 and up to about 3000 amino acid residues. Thus, the XTENinserted into or linked to FVIII or VWF can have lengths of about 6,about 12, about 36, about 40, about 42, about 72, about 96, about 144,about 288, about 400, about 500, about 576, about 600, about 700, about800, about 864, about 900, about 1000, about 1500, about 2000, about2500, or up to about 3000 amino acid residues in length. In otherembodiments, the XTEN sequences is about 6 to about 50, about 50 toabout 100, about 100 to 150, about 150 to 250, about 250 to 400, about400 to about 500, about 500 to about 900, about 900 to 1500, about 1500to 2000, or about 2000 to about 3000 amino acid residues in length. Theprecise length of an XTEN inserted into or linked to FVIII or VWF canvary without adversely affecting the activity of the FVIII or VWF. Inone embodiment, one or more of the XTEN used herein has 36 amino acids,42 amino acids, 72 amino acids, 144 amino acids, 288 amino acids, 576amino acids, or 864 amino acids in length and can be selected from oneor more of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BCor BD.

In some embodiments, the XTEN sequence used in the invention is at least60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to a sequence selected from the group consisting of AE42,AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144, AE180,AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG324, AE360,AG360, AE396, AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504,AE540, AG540, AF540, AD576, AE576, AF576, AG576, AE612, AG612, AE624,AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828,AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM1318, BC864,BD864, AE948, AE1044, AE1140, AE1236, AE1332, AE1428, AE1524, AE1620,AE1716, AE1812, AE1908, AE2004A, AG948, AG1044, AG1140, AG1236, AG1332,AG1428, AG1524, AG1620, AG1716, AG1812, AG1908, and AG2004. See US2010-0239554 A1.

In one embodiment, the XTEN sequence is at least 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequenceselected from the group consisting of AE42 (SEQ ID NO: 36), AE72 (SEQ IDNO: 127), AE144_2A (SEQ ID NO: 128), AE144_3B (SEQ ID NO: 129), AE144_4A(SEQ ID NO: 130), AE144_5A (SEQ ID NO: 131), AE144_6B (SEQ ID NO: 132),AG144_A (SEQ ID NO: 133), AG144_B (SEQ ID NO: 134), AG144_C (SEQ ID NO:135), AG144_F (SEQ ID NO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO:41), AE288 (SEQ ID NO: 39), AE288_2 (SEQ ID NO: 137), AE144 (SEQ ID NO:37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO:40), AG144 (SEQ ID NO: 38), and any combinations thereof.

In some embodiments, less than 100% of amino acids of an XTEN areselected from glycine (G), alanine (A), serine (S), threonine (T),glutamate (E) and proline (P), or less than 100% of the sequenceconsists of the sequence motifs from Table 2A or the XTEN sequences ofTable 2B. In such embodiments, the remaining amino acid residues of theXTEN are selected from any of the other 14 natural L-amino acids, butmay be preferentially selected from hydrophilic amino acids such thatthe XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or at least about 99% hydrophilic amino acids. Thecontent of hydrophobic amino acids in the XTEN utilized in theconjugation constructs may be less than 5%, or less than 2%, or lessthan 1% hydrophobic amino acid content. Hydrophobic residues that areless favored in construction of XTEN include tryptophan, phenylalanine,tyrosine, leucine, isoleucine, valine, and methionine. Additionally,XTEN sequences may contain less than 5% or less than 4% or less than 3%or less than 2% or less than 1% or none of the following amino acids:methionine (for example, to avoid oxidation), or asparagine andglutamine (to avoid desamidation).

In another embodiment, the XTEN sequence is selected from the groupconsisting of AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144_2A (SEQID NO: 128), AE144_3B (SEQ ID NO: 129), AE144_4A (SEQ ID NO: 130),AE144_5A (SEQ ID NO: 131), AE144_6B (SEQ ID NO: 132), AG144_A (SEQ IDNO: 133), AG144_B (SEQ ID NO: 134), AG144_C (SEQ ID NO: 135), AG144_F(SEQ ID NO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288(SEQ ID NO: 39), AE288_2 (SEQ ID NO: 137), AE144 (SEQ ID NO: 37), AG864(SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO: 40), AG144(SEQ ID NO: 38), and any combinations thereof. In a specific embodiment,the XTEN sequence is AE288. The amino acid sequences for certain XTENsequences of the invention are shown in Table 2B.

TABLE 2B XTEN Sequences XTEN Amino Acid Sequence AE42GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS SEQ ID NO: 36 AE72GAPTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA SEQ ID NO:TSGSETPGTSESATPESGPGTSTEPSEGSAPGASS 127 AE144GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTE SEQ ID EGTSTEPSEG NO: 37SAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSE GSAPGTSESAPESGPGSEPATSGSETPGTSTEPSEGSAP AE144_2ATSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP (SEQ ID NO:GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET 128)PGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES GPG AE144_3BSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP (SEQ ID NO:GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA 129)PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS APG AE144_4ATSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP (SEQ ID NO:GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE 130)EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APG AE144_5ATSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP (SEQ ID NO:GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESG 131)PGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST EEG AE144_6BTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP (SEQ ID NO:GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSA 132)PGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APG AG144GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG SEQ ID PGASPGTSST NO: 38GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS TGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSP AG144_AGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSS (SEQ ID NO:PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASS 133)SPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST GSP AG144_BGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSS (SEQ ID NO:PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATG 134)SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST GSP AG144_CGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS (SEQ ID NO:PGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG 135)SPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST GSP AG144_FGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS (SEQ ID NO:PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT 136)GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST GSP AE288GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET SEQ ID PPGTSESATPESGNO: 39 GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAP AE288_2GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG (SEQ ID NO:PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS 137)APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAP AG288PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG SEQ ID SPGTPGSGTASSNO: 40 SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG ATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPS GATGS AE576GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE SEQ ID EGTSTEPSEGSANO: 41 PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AG576PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT SEQ ID GPGSSTPSGATGNO: 42 SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST GSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAST GTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG ATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS GATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPG SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGT PGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS AE864GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE SEQ ID EGTSTEPSEGSANO: 43 PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS APGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AG864GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSS SEQ ID PGSSTPSGATGSNO: 44 PGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG SPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSA STGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTP SGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSST PSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGT PGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS PGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP

In further embodiments, the XTEN sequence used in the invention affectsthe physical or chemical property, e.g., pharmacokinetics, of thechimeric protein of the present invention. The XTEN sequence used in thepresent invention can exhibit one or more of the following advantageousproperties: conformational flexibility, enhanced aqueous solubility,high degree of protease resistance, low immunogenicity, low binding tomammalian receptors, or increased hydrodynamic (or Stokes) radii. In aspecific embodiment, the XTEN sequence linked to a FVIII protein in thisinvention increases pharmacokinetic properties such as longer terminalhalf-life or increased area under the curve (AUC), so that the chimericprotein described herein stays in vivo for an increased period of timecompared to wild type FVIII. In further embodiments, the XTEN sequenceused in this invention increases pharmacokinetic properties such aslonger terminal half-life or increased area under the curve (AUC), sothat FVIII protein stays in vivo for an increased period of timecompared to wild type FVIII.

A variety of methods and assays can be employed to determine thephysical/chemical properties of proteins comprising the XTEN sequence.Such methods include, but are not limited to analytical centrifugation,EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, lightscattering, capillary electrophoresis, circular dichroism, differentialscanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-sizeexclusion, IR, NMR, Raman spectroscopy, refractometry, and UV/Visiblespectroscopy. Additional methods are disclosed in Amau et al., Prot Exprand Purif 48, 1-13 (2006).

Additional examples of XTEN sequences that can be used according to thepresent invention and are disclosed in US Patent Publication Nos.2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1,2011/0077199 A1, or 2011/0172146 A1, or International Patent PublicationNos. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228A1, WO 2011028229 A1, or WO 2011028344 A2.

C) Factor VIII (FVIII) Protein

“A FVIII protein” as used herein means a functional FVIII polypeptide inits normal role in coagulation, unless otherwise specified. The term aFVIII protein includes a functional fragment, variant, analog, orderivative thereof that retains the function of full-length wild-typeFactor VIII in the coagulation pathway. “A FVIII protein” is usedinterchangeably with FVIII polypeptide (or protein) or FVIII. Examplesof the FVIII functions include, but not limited to, an ability toactivate coagulation, an ability to act as a cofactor for factor IX, oran ability to form a tenase complex with factor IX in the presence ofCa′ and phospholipids, which then converts Factor X to the activatedform Xa. The FVIII protein can be the human, porcine, canine, rat, ormurine FVIII protein. In addition, comparisons between FVIII from humansand other species have identified conserved residues that are likely tobe required for function (Cameron et al., Thromb. Haemost. 79:317-22(1998); U.S. Pat. No. 6,251,632).

A number of tests are available to assess the function of thecoagulation system: activated partial thromboplastin time (aPTT) test,chromogenic assay, ROTEM assay, prothrombin time (PT) test (also used todetermine INR), fibrinogen testing (often by the Clauss method),platelet count, platelet function testing (often by PFA-100), TCT,bleeding time, mixing test (whether an abnormality corrects if thepatient's plasma is mixed with normal plasma), coagulation factorassays, antiphospholipid antibodies, D-dimer, genetic tests (e.g.,factor V Leiden, prothrombin mutation G20210A), dilute Russell's vipervenom time (dRVVT), miscellaneous platelet function tests,thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g.,ROTEM®) or euglobulin lysis time (ELT).

The aPTT test is a performance indicator measuring the efficacy of boththe “intrinsic” (also referred to the contact activation pathway) andthe common coagulation pathways. This test is commonly used to measureclotting activity of commercially available recombinant clottingfactors, e.g., FVIII or FIX. It is used in conjunction with prothrombintime (PT), which measures the extrinsic pathway.

ROTEM analysis provides information on the whole kinetics ofhaemostasis: clotting time, clot formation, clot stability and lysis.The different parameters in thromboelastometry are dependent on theactivity of the plasmatic coagulation system, platelet function,fibrinolysis, or many factors which influence these interactions. Thisassay can provide a complete view of secondary haemostasis.

The FVIII polypeptide and polynucleotide sequences are known, as aremany functional fragments, mutants and modified versions. Examples ofhuman FVIII sequences (full-length) are shown below.

TABLE 3 Amino Acid Sequence of Full-length Factor VIII(Full-length FVIII (FVIII signal peptide underlined; FVIII heavychain is double underlined; B domain is italicized; and FVIIIlight chain is in plain text) Signal Peptide: (SEQ ID NO: 3)MQIELSTCFFLCLLRFCFS Mature Factor VIII (SEQ ID NO: 4)*ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPR SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMIMDKNATALRENHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMIFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNIFLTNIDNIHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNIFLLSTRQNVEGSYDGAYAPVLQDFRSENDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTIIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIKETRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNISLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVITQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY

TABLE 4 Nucleotide Sequence Encoding Full-Length FVIII (SEQ ID NO: 5)* 661                                         ATG CAAATAGAGC TCTCCACCTG 721 CTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACC AGAAGATACT ACCTGGGTGC 781 AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGT GAGCTGCCTG TGGACGCAAG 841 ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC TCAGTCGTGT ACAAAAAGAC 901 TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT AAGCCAAGGC CACCCTGGAT 961 GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGAT ACAGTGGTCA TTACACTTAA1021 GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGGT GTATCCTACT GGAAAGCTTC1081 TGAGGGAGCT GAATATGATG ATCAGACCAG TCAAAGGGAG AAAGAAGATG ATAAAGTCTT1141 CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA GAGAATGGTC CAATGGCCTC1201 TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG GACCTGGTAA AAGACTTGAA1261 TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGG AGTCTGGCCA AGGAAAAGAC1321 ACAGACCTTG CACAAATTTA TACTACTTTT TGCTGTATTT GATGAAGGGA AAAGTTGGCA1381 CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCT GCATCTGCTC GGGCCTGGCC1441 TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTG CCAGGTCTGA TTGGATGCCA1501 CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC ACTCCTGAAG TGCACTCAAT1561 ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGC CAGGCGTCCT TGGAAATCTC1621 GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGAC CTTGGACAGT TTCTACTGTT1681 TTGTCATATC TCTTCCCACC AACATGATGG CATGGAAGCT TATGTCAAAG TAGACAGCTG1741 TCCAGAGGAA CCCCAACTAC GAATGAAAAA TAATGAAGAA GCGGAAGACT ATGATGATGA1801 TCTTACTGAT TCTGAAATGG ATGTGGTCAG GTTTGATGAT GACAACTCTC CTTCCTTTAT1861 CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGG GTACATTACA TTGCTGCTGA1921 AGAGGAGGAC TGGGACTATG CTCCCTTAGT CCTCGCCCCC GATGACAGAA GTTATAAAAG1981 TCAATATTTG AACAATGGCC CTCAGCGGAT TGGTAGGAAG TACAAAAAAG TCCGATTTAT2041 GGCATACACA GATGAAACCT TTAAGACTCG TGAAGCTATT CAGCATGAAT CAGGAATCTT2101 GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTGTTG ATTATATTTA AGAATCAAGC2161 AAGCAGACCA TATAACATCT ACCCTCACGG AATCACTGAT GTCCGTCCTT TGTATTCAAG2221 GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCA ATTCTGCCAG GAGAAATATT2281 CAAATATAAA TGGACAGTGA CTGTAGAAGA TGGGCCAACT AAATCAGATC CTCGGTGCCT2341 GACCCGCTAT TACTCTAGTT TCGTTAATAT GGAGAGAGAT CTAGCTTCAG GACTCATTGG2401 CCCTCTCCTC ATCTGCTACA AAGAATCTGT AGATCAAAGA GGAAACCAGA TAATGTCAGA2461 CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAAC CGAAGCTGGT ACCTCACAGA2521 GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAG CTTGAGGATC CAGAGTTCCA2581 AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTT GATAGTTTGC AGTTGTCAGT2641 TTGTTTGCAT GAGGTGGCAT ACTGGTACAT TCTAAGCATT GGAGCACAGA CTGACTTCCT2701 TTCTGTCTTC TTCTCTGGAT ATACCTTCAA ACACAAAATG GTCTATGAAG ACACACTCAC2761 CCTATTCCCA TTCTCAGGAG AAACTGTCTT CATGTCGATG GAAAACCCAG GTCTATGGAT2821 TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATG ACCGCCTTAC TGAAGGTTTC2881 TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGT TATGAAGATA TTTCAGCATA2941 CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTC TCCCAGAATT CAAGACACCC3001 TAGCACTAGG CAAAAGCAAT TTAATGCCAC CACAATTCCA GAAAATGACA TAGAGAAGAC3061 TGACCCTTGG TTTGCACACA GAACACCTAT GCCTAAAATA CAAAATGTCT CCTCTAGTGA3121 TTTGTTGATG CTCTTGCGAC AGAGTCCTAC TCCACATGGG CTATCCTTAT CTGATCTCCA3181 AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCT GGAGCAATAG ACAGTAATAA3241 CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCAT CACAGTGGGG ACATGGTATT3301 TACCCCTGAG TCAGGCCTCC AATTAAGATT AAATGAGAAA CTGGGGACAA CTGCAGCAAC3361 AGAGTTGAAG AAACTTGATT TCAAAGTTTC TAGTACATCA AATAATCTGA TTTCAACAAT3421 TCCATCAGAC AATTTGGCAG CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT3481 GCCAGTTCAT TATGATAGTC AATTAGATAC CACTCTATTT GGCAAAAAGT CATCTCCCCT3541 TACTGAGTCT GGTGGACCTC TGAGCTTGAG TGAAGAAAAT AATGATTCAA AGTTGTTAGA3601 ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAA AATGTATCGT CAACAGAGAG3661 TGGTAGGTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCT TTGTTGACTA AAGATAATGC3721 CTTATTCAAA GTTAGCATCT CTTTGTTAAA GACAAACAAA ACTTCCAATA ATTCAGCAAC3781 TAATAGAAAG ACTCACATTG ATGGCCCATC ATTATTAATT GAGAATAGTC CATCAGTCTG3841 GCAAAATATA TTAGAAAGTG ACACTGAGTT TAAAAAAGTG ACACCTTTGA TTCATGACAG3901 AATGCTTATG GACAAAAATG CTACAGCTTT GAGGCTAAAT CATATGTCAA ATAAAACTAC3961 TTCATCAAAA AACATGGAAA TGGTCCAACA GAAAAAAGAG GGCCCCATTC CACCAGATGC4021 ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTG CCAGAATCAG CAAGGTGGAT4081 ACAAAGGACT CATGGAAAGA ACTCTCTGAA CTCTGGGCAA GGCCCCAGTC CAAAGCAATT4141 AGTATCCTTA GGACCAGAAA AATCTGTGGA AGGTCAGAAT TTCTTGTCTG AGAAAAACAA4201 AGTGGTAGTA GGAAAGGGTG AATTTACAAA GGACGTAGGA CTCAAAGAGA TGGTTTTTCC4261 AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTA CATGAAAATA ATACACACAA4321 TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAA ACATTAATCC AAGAGAATGT4381 AGTTTTGCCT CAGATACATA CAGTGACTGG CACTAAGAAT TTCATGAAGA ACCTTTTCTT4441 ACTGAGCACT AGGCAAAATG TAGAAGGTTC ATATGACGGG GCATATGCTC CAGTACTTCA4501 AGATTTTAGG TCATTAAATG ATTCAACAAA TAGAACAAAG AAACACACAG CTCATTTCTC4561 AAAAAAAGGG GAGGAAGAAA ACTTGGAAGG CTTGGGAAAT CAAACCAAGC AAATTGTAGA4621 GAAATATGCA TGCACCACAA GGATATCTCC TAATACAAGC CAGCAGAATT TTGTCACGCA4681 ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTA GAAGAAACAG AACTTGAAAA4741 AAGGATAATT GTGGATGACA CCTCAACCCA GTGGTCCAAA AACATGAAAC ATTTGACCCC4801 GAGCACCCTC ACACAGATAG ACTACAATGA GAAGGAGAAA GGGGCCATTA CTCAGTCTCC4861 CTTATCAGAT TGCCTTACGA GGAGTCATAG CATCCCTCAA GCAAATAGAT CTCCATTACC4921 CATTGCAAAG GTATCATCAT TTCCATCTAT TAGACCTATA TATCTGACCA GGGTCCTATT4981 CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGA AAGAAAGATT CTGGGGTCCA5041 AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAAC CTTTCTTTAG CCATTCTAAC5101 CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTG GGGACAAGTG CCACAAATTC5161 AGTCACATAC AAGAAAGTTG AGAACACTGT TCTCCCGAAA CCAGACTTGC CCAAAACATC5221 TGGCAAAGTT GAATTGCTTC CAAAAGTTCA CATTTATCAG AAGGACCTAT TCCCTACGGA5281 AACTAGCAAT GGGTCTCCTG GCCATCTGGA TCTCGTGGAA GGGAGCCTTC TTCAGGGAAC5341 AGAGGGAGCG ATTAAGTGGA ATGAAGCAAA CAGACCTGGA AAAGTTCCCT TTCTGAGAGT5401 AGCAACAGAA AGCTCTGCAA AGACTCCCTC CAAGCTATTG GATCCTCTTG CTTGGGATAA5461 CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCC CAAGAGAAGT CACCAGAAAA5521 AACAGCTTTT AAGAAAAAGG ATACCATTTT GTCCCTGAAC GCTTGTGAAA GCAATCATGC5581 AATAGCAGCA ATAAATGAGG GACAAAATAA GCCCGAAATA GAAGTCACCT GGGCAAAGCA5641 AGGTAGGACT GAAAGGCTGT GCTCTCAAAA CCCACCAGTC TTGAAACGCC ATCAACGGGA5701 AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG ATACCATATC5761 AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC AGAGCCCCCG5821 CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC TCTGGGATTA5881 TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA GTGTCCCTCA5941 GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC CCTTATACCG6001 TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG AAGTTGAAGA6061 TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT ATTCTAGCCT6121 TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT TTGTCAAGCC6181 TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA CTAAAGATGA6241 GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG ATGTGCACTC6301 AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG CTCATGGGAG6361 ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA CCAAAAGCTG6421 GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA6481 TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA TGGATACACT6541 ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA GCATGGGCAG6601 CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC GAAAAAAAGA6661 GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG TGGAAATGTT6721 ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC TACATGCTGG6781 GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG GAATGGCTTC6841 TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT GGGCCCCAAA6901 GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG AGCCCTTTTC6961 TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA CCCAGGGTGC7021 CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA GTCTTGATGG7081 GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT TCTTTGGCAA7141 TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG CTCGATACAT7201 CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT TGATGGGCTG7261 TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT CAGATGCACA7321 GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT CAAAAGCTCG7381 ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC CAAAAGAGTG7441 GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC AGGGAGTAAA7501 ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC AAGATGGCCA7561 TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA ATCAAGACTC7621 CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC TTCGAATTCA7681 CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT GCGAGGCACA7741 GGACCTCTAC *The underlined nucleic acids encode a signal peptide.

FVIII polypeptides include full-length FVIII, full-length FVIII minusMet at the N-terminus, mature FVIII (minus the signal sequence), matureFVIII with an additional Met at the N-terminus, and/or FVIII with a fullor partial deletion of the B domain. In certain embodiments, FVIIIvariants include B domain deletions, whether partial or full deletions.

The sequence of native mature human FVIII is presented as SEQ ID NO: 4.A native FVIII protein has the following formula:A1-a1-A2-a2-B-a3-A3-C1-C2, where A1, A2, and A3 are thestructurally-related “A domains,” B is the “B domain,” C1 and C2 are thestructurally-related “C domains,” and a1, a2 and a3 are acidic spacerregions. Referring to the primary amino acid sequence position in SEQ IDNO:4, the A1 domain of human FVIII extends from Ala1 to about Arg336,the a1 spacer region extends from about Met337 to about Val374, the A2domain extends from about Ala375 to about Tyr719, the a2 spacer regionextends from about Glu720 to about Arg740, the B domain extends fromabout Ser741 to about Arg 1648, the a3 spacer region extends from aboutGlu1649 to about Arg1689, the A3 domain extends from about Ser1690 toabout Leu2025, the C1 domain extends from about Gly2026 to aboutAsn2072, and the C2 domain extends from about Ser2073 to Tyr2332. Otherthan specific proteolytic cleavage sites, designation of the locationsof the boundaries between the domains and regions of FVIII can vary indifferent literature references. The boundaries noted herein aretherefore designated as approximate by use of the term “about.”

The human FVIII gene was isolated and expressed in mammalian cells(Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al.,Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337(1984); Vehar, G. A., et al., Nature 312:337-342 (1984); WO 87/04187; WO88/08035; WO 88/03558; and U.S. Pat. No. 4,757,006). The FVIII aminoacid sequence was deduced from cDNA as shown in U.S. Pat. No. 4,965,199.In addition, partially or fully B-domain deleted FVIII is shown in U.S.Pat. Nos. 4,994,371 and 4,868,112. In some embodiments, the human FVIIIB-domain is replaced with the human Factor V B-domain as shown in U.S.Pat. No. 5,004,803. The cDNA sequence encoding human Factor VIII andamino acid sequence are shown in SEQ ID NOs: 4 and 5, respectively, ofUS Application Publ. No. 2005/0100990.

The porcine FVIII sequence is published in Toole, J. J., et al., Proc.Natl. Acad. Sci. USA 83:5939-5942 (1986). Further, the complete porcinecDNA sequence obtained from PCR amplification of FVIII sequences from apig spleen cDNA library has been reported in Healey, J. F., et al.,Blood 88:4209-4214 (1996). Hybrid human/porcine FVIII havingsubstitutions of all domains, all subunits, and specific amino acidsequences were disclosed in U.S. Pat. No. 5,364,771 by Lollar and Runge,and in WO 93/20093. More recently, the nucleotide and correspondingamino acid sequences of the A1 and A2 domains of porcine FVIII and achimeric FVIII with porcine A1 and/or A2 domains substituted for thecorresponding human domains were reported in WO 94/11503. U.S. Pat. No.5,859,204, Lollar, J. S., also discloses the porcine cDNA and deducedamino acid sequences. U.S. Pat. No. 6,458,563 discloses aB-domain-deleted porcine FVIII.

U.S. Pat. No. 5,859,204 to Lollar, J. S. reports functional mutants ofFVIII having reduced antigenicity and reduced immunoreactivity. U.S.Pat. No. 6,376,463 to Lollar, J. S. also reports mutants of FVIII havingreduced immunoreactivity. US Appl. Publ. No. 2005/0100990 to Saenko etal. reports functional mutations in the A2 domain of FVIII.

In one embodiment, the FVIII (or FVIII portion of a chimeric protein)may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to a FVIII amino acid sequence of amino acids 1 to 1438of SEQ ID NO: 6 or amino acids 1 to 2332 of SEQ ID NO: 4 (without asignal sequence) or a FVIII amino acid sequence of amino acids 1 to 19of SEQ ID NO: 3 and 1 to 1438 of SEQ ID NO: 6 or amino acids 1 to 19 ofSEQ ID NO: 3 and amino acids 1 to 2332 of SEQ ID NO: 4 (with a signalsequence), wherein the FVIII has a clotting activity, e.g., activatesFactor IX as a cofactor to convert Factor X to activated Factor X. TheFVIII (or FVIII portion of a chimeric protein) may be identical to aFVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 6 oramino acids 1 to 2332 of SEQ ID NO: 4 (without a signal sequence). TheFVIII may further comprise a signal sequence.

The “B-domain” of FVIII, as used herein, is the same as the B-domainknown in the art that is defined by internal amino acid sequenceidentity and sites of proteolytic cleavage, e.g., residuesSer741-Arg1648 of full-length human FVIII. The other human FVIII domainsare defined by the following amino acid residues: A1, residuesAla1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Asn2019;C1, residues Lys2020-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2sequence includes residues Ser1690-Tyr2332. The remaining sequence,residues Glu1649-Arg1689, is usually referred to as the a3 acidicregion. The locations of the boundaries for all of the domains,including the B-domains, for porcine, mouse and canine FVIII are alsoknown in the art. In one embodiment, the B domain of FVIII is deleted(“B-domain-deleted factor VIII” or “BDD FVIII”). An example of a BDDFVIII is REFACTO® (recombinant BDD FVIII), which has the same sequenceas the Factor VIII portion of the sequence in Table 5. (BDD FVIII heavychain is double underlined; B domain is italicized; and BDD FVIII lightchain is in plain text). A nucleotide sequence encoding the amino acidsequence set forth in Table 5 (SEQ ID NO: 7) is shown in Table 6.

TABLE 5 Amino Acid Sequence of B-domain Deleted Factor VIII (BDD FVIII)BDD FVIII (SEQ ID NO: 6)ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPR SFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVITQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVL GCEAQDLY

TABLE 6 Nucleotide Sequence Encoding BDD FVIII (SEQ ID NO: 7)*  661                               A TGCAAATAGA GCTCTCCACC TGCTTCTTTC  721TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA CTACCTGGGT GCAGTGGAAC  781TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCC TGTGGACGCA AGATTTCCTC  841CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGT GTACAAAAAG ACTCTGTTTG  901TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC  961TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT AAGAACATGG 1021CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA CTGGAAAGCT TCTGAGGGAG 1081CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGA TGATAAAGTC TTCCCTGGTG 1141GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATGG TCCAATGGCC TCTGACCCAC 1201TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC 1261TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT 1321TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG GAAAAGTTGG CACTCAGAAA 1381CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTGC TCGGGCCTGG CCTAAAATGC 1441ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT GATTGGATGC CACAGGAAAT 1501CAGTCTATTG GCATGTGATT GGAATGGGCA CCACTCCTGA AGTGCACTCA ATATTCCTCG 1561AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA 1621CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA GTTTCTACTG TTTTGTCATA 1681TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA AGTAGACAGC TGTCCAGAGG 1741AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGA CTATGATGAT GATCTTACTG 1801ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTC TCCTTCCTTT ATCCAAATTC 1861GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA CATTGCTGCT GAAGAGGAGG 1921ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG AAGTTATAAA AGTCAATATT 1981TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAA AGTCCGATTT ATGGCATACA 2041CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGA ATCAGGAATC TTGGGACCTT 2101TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA GCAAGCAGAC 2161CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC TTTGTATTCA AGGAGATTAC 2221CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC AGGAGAAATA TTCAAATATA 2281AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCGCT 2341ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GGCCCTCTCC 2401TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA GATAATGTCA GACAAGAGGA 2461ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTG GTACCTCACA GAGAATATAC 2521AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA TCCAGAGTTC CAAGCCTCCA 2581ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT GCAGTTGTCA GTTTGTTTGC 2641ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT 2701TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC 2761CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCC AGGTCTATGG ATTCTGGGGT 2821GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT ACTGAAGGTT TCTAGTTGTG 2881ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA 2941GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA CCCACCAGTC TTGAAACGCC 3001ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG 3061ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC 3121AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC 3181TCTGGGATTA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA 3241GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC 3301CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG 3361AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT 3421ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT 3481TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA 3541CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG 3601ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG 3661CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA 3721CCAAAAGCTG GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC 3781AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA 3841TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA 3901GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC 3961GAAAAAAAGA GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG 4021TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC 4081TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG 4141GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT 4201GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG 4261AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA 4321CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA 4381GTCTTGATGG GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT 4441TCTTTGGCAA TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG 4501CTCGATACAT CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT 4561TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT 4621CAGATGCACA GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT 4681CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC 4741CAAAAGAGTG GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC 4801AGGGAGTAAA ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC 4861AAGATGGCCA TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA 4921ATCAAGACTC CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC 4981TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT 5041GCGAGGCACA GGACCTCTAC *The underlined nucleic acids encode a signalpeptide.

A “B-domain-deleted FVIII” may have the full or partial deletionsdisclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203,6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502,5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563. In someembodiments, a B-domain-deleted FVIII sequence of the present inventioncomprises any one of the deletions disclosed at col. 4, line 4 to col.5, line 28 and Examples 1-5 of U.S. Pat. No. 6,316,226 (also in U.S.Pat. No. 6,346,513). In another embodiment, a B-domain deleted FactorVIII is the S743/Q1638 B-domain deleted Factor VIII (SQ BDD FVIII)(e.g., Factor VIII having a deletion from amino acid 744 to amino acid1637, e.g., Factor VIII having amino acids 1-743 and amino acids1638-2332 of SEQ ID NO: 4, i.e., SEQ ID NO: 6). In some embodiments, aB-domain-deleted FVIII of the present invention has a deletion disclosedat col. 2, lines 26-51 and examples 5-8 of U.S. Pat. No. 5,789,203 (alsoU.S. Pat. Nos. 6,060,447, 5,595,886, and 6,228,620). In someembodiments, a B-domain-deleted Factor VIII has a deletion described incol. 1, lines 25 to col. 2, line 40 of U.S. Pat. No. 5,972,885; col. 6,lines 1-22 and example 1 of U.S. Pat. No. 6,048,720; col. 2, lines 17-46of U.S. Pat. No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S.Pat. No. 5,171,844; col. 2, lines 55-68, FIG. 2, and example 1 of U.S.Pat. No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 ofU.S. Pat. No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11,line 5 to col. 13, line 39 of U.S. Pat. No. 7,041,635; or col. 4, lines25-53, of U.S. Pat. No. 6,458,563. In some embodiments, aB-domain-deleted FVIII has a deletion of most of the B domain, but stillcontains amino-terminal sequences of the B domain that are essential forin vivo proteolytic processing of the primary translation product intotwo polypeptide chain, as disclosed in WO 91/09122. In some embodiments,a B-domain-deleted FVIII is constructed with a deletion of amino acids747-1638, i.e., virtually a complete deletion of the B domain. Hoeben R.C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990). A B-domain-deletedFactor VIII may also contain a deletion of amino acids 771-1666 or aminoacids 868-1562 of FVIII. Meulien P., et al. Protein Eng. 2(4): 301-6(1988). Additional B domain deletions that are part of the inventioninclude: deletion of amino acids 982 through 1562 or 760 through 1639(Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797through 1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), 741through 1646 (Kaufman (PCT published application No. WO 87/04187)),747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741 through 1648(Pasek (PCT application No. 88/00831)), or 816 through 1598 or 741through 1648 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP295597)). In other embodiments, BDD FVIII includes a FVIII polypeptidecontaining fragments of the B-domain that retain one or more N-linkedglycosylation sites, e.g., residues 757, 784, 828, 900, 963, oroptionally 943, which correspond to the amino acid sequence of thefull-length FVIII sequence. Examples of the B-domain fragments include226 amino acids or 163 amino acids of the B-domain as disclosed in Miao,H. Z., et al., Blood 103(a): 3412-3419 (2004), Kasuda, A, et al., J.Thromb. Haemost. 6: 1352-1359 (2008), and Pipe, S. W., et al., J.Thromb. Haemost. 9: 2235-2242 (2011) (i.e., the first 226 amino acids or163 amino acids of the B domain are retained). In still otherembodiments, BDD FVIII further comprises a point mutation at residue 309(from Phe to Ser) to improve expression of the BDD FVIII protein. SeeMiao, H. Z., et al., Blood 103(a): 3412-3419 (2004). In still otherembodiments, the BDD FVIII includes a FVIII polypeptide containing aportion of the B-domain, but not containing one or more furin cleavagesites (e.g., Arg1313 and Arg 1648). See Pipe, S. W., et al., J. Thromb.Haemost. 9: 2235-2242 (2011). Each of the foregoing deletions may bemade in any FVIII sequence.

In some embodiments, the FVIII has a partial B-domain. In someembodiments, the FVIII protein with a partial B-domain is FVIII198 (SEQID NO: 89). FVIII198 is a partial B-domain containing single chainFVIIIFc molecule-226N6. 226 represents the N-terminus 226 amino acid ofthe FVIII B-domain, and N6 represents six N-glycosylation sites in theB-domain.

In one embodiment, FVIII is cleaved right after arginine at amino acid1648 (in full-length Factor VIII or SEQ ID NO: 4), amino acid 754 (inthe 5743/Q1638 B-domain deleted Factor VIII or SEQ ID NO: 6), or thecorresponding arginine residue (in other variants), thereby resulting ina heavy chain and a light chain. In another embodiment, FVIII comprisesa heavy chain and a light chain, which are linked or associated by ametal ion-mediated non-covalent bond.

In other embodiments, FVIII is a single chain FVIII that has not beencleaved right after Arginine at amino acid 1648 (in full-length FVIII orSEQ ID NO: 4), amino acid 754 (in the 5743/Q1638 B-domain-deleted FVIIIor SEQ ID NO: 6), or the corresponding Arginine residue (in othervariants). A single chain FVIII may comprise one or more amino acidsubstitutions. In one embodiment, the amino acid substitution is at aresidue corresponding to residue 1648, residue 1645, or both offull-length mature Factor VIII polypeptide (SEQ ID NO: 4) or residue754, residue 751, or both of SQ BDD Factor VIII (SEQ ID NO: 6). Theamino acid substitution can be any amino acids other than arginine,e.g., isoleucine, leucine, lysine, methionine, phenylalanine, threonine,tryptophan, valine, alanine, asparagine, aspartic acid, cysteine,glutamic acid, glutamine, glycine, proline, selenocysteine, serine,tyrosine, histidine, ornithine, pyrrolysine, or taurine.

FVIII can further be cleaved by thrombin and then activated as FVIIIa,serving as a cofactor for activated Factor IX (FIXa). And the activatedFIX together with activated FVIII forms a Xase complex and convertsFactor X to activated Factor X (FXa). For activation, FVIII is cleavedby thrombin after three Arginine residues, at amino acids 372, 740, and1689 (corresponding to amino acids 372, 740, and 795 in the B-domaindeleted FVIII sequence), the cleavage generating FVIIIa having the 50kDa A1, 43 kDa A2, and 73 kDa A3-C1-C2 chains. In one embodiment, theFVIII protein useful for the present invention is non-active FVIII. Inanother embodiment, the FVIII protein is an activated FVIII.

The protein having FVIII polypeptide linked to or associated with theVWF fragment can comprise a sequence at least 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or 6, whereinthe sequence has the FVIII clotting activity, e.g., activating Factor IXas a cofactor to convert Factor X to activated Factor X (FXa).

“Hybrid” or “chimeric” polypeptides and proteins, as used herein,includes a combination of a first polypeptide chain, e.g., the VWFfragment, optionally fused to a first Ig constant region or a portionthereof, with a second polypeptide chain, e.g., a FVIII protein linkedto an XTEN sequence, optionally fused to a second Ig constant region ora portion thereof, thereby forming a heterodimer. In one embodiment, thefirst polypeptide and the second polypeptide in a hybrid are associatedwith each other via protein-protein interactions, such as charge-chargeor hydrophobic interactions. In another embodiment, the firstpolypeptide and the second polypeptide in a hybrid are associated witheach other via disulfide or other covalent bond(s). Hybrids aredescribed, for example, in US 2004/101740 and US 2006/074199. The secondpolypeptide may be an identical copy of the first polypeptide or anon-identical polypeptide. In one embodiment, the first polypeptide is aFVIII protein(X)-Fc fusion protein, and the second polypeptide is apolypeptide comprising, consisting essentially of, or consisting of anFc region, wherein the first polypeptide and the second polypeptide areassociated with each other. In another embodiment, the first polypeptidecomprises a VWF fragment-XTEN-Fc fusion protein, and the secondpolypeptide comprises FVIII-Fc fusion protein, making the hybrid aheterodimer. In other embodiments, the first polypeptide comprises a VWFfragment-Fc fusion protein, and the second polypeptide comprisesFVIII(X)-Fc fusion protein, making the hybrid a heterodimer. In yetother embodiments, the first polypeptide comprises a VWFfragment-XTEN-Fc fusion protein, and the second polypeptide comprisesFVIII(X)-Fc fusion protein. The first polypeptide and the secondpolypeptide can be associated through a covalent bond, e.g., a disulfidebond, between the first Fc region and the second Fc region. The firstpolypeptide and the second polypeptide can further be associated witheach other by binding between the VWF fragment and the FVIII protein.

A FVIII protein useful in the present invention can include FVIII havingone or more additional XTEN sequences, which do not affect the FVIIIcoagulation activity. Such XTEN sequences can be fused to the C-terminusor N-terminus of the FVIII protein or inserted between one or more ofthe two amino acid residues in the FVIII protein wherein the insertionsdo not affect the FVIII coagulation activity or FVIII function. In oneembodiment, the insertions improve pharmacokinetic properties of theFVIII protein (e.g., half-life). In another embodiment, the insertionscan be multiple insertions, e.g., more than two, three, four, five, six,seven, eight, nine, or ten insertions. Examples of the insertion sitesinclude, but are not limited to, the sites listed in Tables 7, 8, 9, 10,11, 12, 13, 14, 15 or any combinations thereof.

The FVIII protein linked to one or more XTEN sequences can berepresented as FVIII(X), FVIII(X1), FVIII_((a→b))-X-FVIII_((c→d)),wherein FVIII_((a→b)) comprises, consists essentially of, or consists ofa first portion of a FVIII protein from amino acid residue “a” to aminoacid residue “b”; X or X1 comprises, consists essentially of, orconsists of one or more XTEN sequences, FVIII_((c→d)) comprises,consists essentially of, or consists of a second portion of a FVIIIprotein from amino acid residue “c” to amino acid residue “d”; a is theN-terminal amino acid residue of the first portion of the FVIII protein,b is the C-terminal amino acid residue of the first portion of the FVIIIprotein but is also the N-terminal amino acid residue of the two aminoacids of an insertion site in which the XTEN sequence is inserted, c isthe N-terminal amino acid residue of the second portion of the FVIIIprotein but is also the C-terminal amino acid residue of the two aminoacids of an insertion site in which the XTEN sequence is inserted, and dis the C-terminal amino acid residue of the FVIII protein, and whereinthe first portion of the FVIII protein and the second portion of theFVIII protein are not identical to each other and are of sufficientlength together such that the FVIII protein has a FVIII coagulationactivity.

In one embodiment, the first portion of the FVIII protein and the secondportion of the FVIII protein are fragments of SEQ ID NO: 4 [full lengthmature FVIII sequence] or SEQ ID NO: 6 [B-domain deleted FVIII], e.g.,N-terminal portion and C-terminal portion, respectively. In certainembodiments, the first portion of the FVIII protein comprises the A1domain and the A2 domain of the FVIII protein. The second portion of theFVIII protein comprises the A3 domain, the C1 domain, and optionally theC2 domain. In yet other embodiments, the first portion of the FVIIIprotein comprises the A1 domain and A2 domain, and the second portion ofthe FVIII protein comprises a portion of the B domain, the A3 domain,the C1 domain, and optionally the C2 domain. In still other embodiments,the first portion of the FVIII protein comprises the A1 domain, A2domain, and a portion of the B domain of the FVIII protein, and thesecond portion of the FVIII protein comprises the A3 domain, the C1domain, and optionally the C2 domain. In still other embodiments, thefirst portion of the FVIII protein comprises the A1 domain, A2 domain,and a first portion of the B domain of the FVIII protein. The secondportion of the FVIII protein comprises a second portion of the B domain,the A3 domain, the C1 domain, and optionally the C2 domain. In someembodiments, the two amino acids (“b” and “c”) can be any one or more ofthe amino acid residues insertion sites shown in Tables 7, 8, 9, 10, 11,12, 13, 14, and 15. For example, “b” can be the amino acid residueimmediately upstream of the site in which one or more XTEN sequences areinserted or linked, and “c” can be the amino acid residue immediatelydownstream of the site in which the one or more XTEN sequences areinserted or linked. In some embodiments, “a” is the first mature aminoacid sequence of a FVIII protein, and “d” is the last amino acidsequence of a FVIII protein. For example, FVIII_((a→b)) can be an aminoacid sequence at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 1 to 745 of SEQ ID NO: 6 [B domain deletedFVIII amino acid sequence] or SEQ ID NO: 4 [full length FVIII] andFVIII_((c→d)) can be amino acids 746 to 1438 of SEQ ID NO: 6 or aminoacids 1641 to 2332 of SEQ ID NO: 4, respectively.

In some aspects, the insertion site in the FVIII protein is located inone or more domains of the FVIII protein, which is the N-terminus, theA1 domain, the A2 domain, the A3 domain, the B domain, the C1 domain,the C2 domain, the C-terminus, or two or more combinations thereof orbetween two domains of the FVIII protein, which are the A1 domain and a1acidic region, and the a1 acidic region and A2 domain, the A2 domain anda2 acidic region, the a2 acidic region and B domain, the B domain and A3domain, and the A3 domain and C1 domain, the C1 domain and C2 domain, orany combinations thereof. For example, the insertion sites in which theXTEN sequence can be inserted are selected from the group consisting ofthe N-terminus and A1 domain, the N-terminus and A2 domain, theN-terminus and A3 domain, the N-terminus and B domain, the N-terminusand C1 domain, the N-terminus and C2 domain, the N-terminus and theC-terminus, the A1 and A2 domains, the A1 and A3 domains, the A1 and Bdomains, the A1 and C1 domains, the A1 and C2 domains, the A1 domain andthe C-terminus, the A2 and A3 domains, the A2 and B domains, the A2 andC1 domains, the A2 and C2 domains, the A2 domain and the C-terminus, theA3 and B domains, the A3 and C1 domains, the A3 and C2 domains, the A3domain and the C-terminus, the B and C1 domains, the B and C2 domains,the B domain and the C-terminus, the C1 and C2 domains, the C1 and theC-terminus, the C2 domain, and the C-terminus, and two or morecombinations thereof. Non-limiting examples of the insertion sites arelisted in Tables 7, 8, 9, 10, 11, 12, 13, 14, and 15.

The FVIII protein, in which the XTEN sequence is inserted immediatelydownstream of one or more amino acids (e.g., one or more XTEN insertionsites) in the FVIII protein or linked at the C-terminus or theN-terminus, retains the FVIII activity after linkage to or insertion bythe XTEN sequence. The XTEN sequence can be inserted in the FVIIIprotein once or more than once, twice, three times, four times, fivetimes, or six times such that the insertions do not affect the FVIIIactivity (i.e., the FVIII protein still retains the coagulationproperty).

The FVIII protein useful in the present invention can be linked to oneor more XTEN polypeptides at the N-terminus or C-terminus of the FVIIIprotein by an optional linker or inserted immediately downstream of oneor more amino acids (e.g., one or more XTEN insertion sites) in theFVIII protein by one or more optional linkers. In one embodiment, thetwo amino acid residues in which the XTEN sequence is inserted or theamino acid residue to which the XTEN sequence is linked correspond tothe two or one amino acid residues of SEQ ID NO: 4 [full length matureFVIII] selected from the group consisting of the residues in Table 7,Table 8, Table 9, and Table 10 and any combinations thereof.

In other embodiments, at least one XTEN sequence is inserted in any oneor more XTEN insertion sites disclosed herein or any combinationsthereof. In one aspect, at least one XTEN sequence is inserted in one ormore XTEN insertion sites disclosed in one or more amino acids disclosedin Table 7.

TABLE 7 Exemplary XTEN Insertion Sites XTEN FVIII BDD InsertionInsertion Downstream FVIII No. Point* Residue Sequence Domain 1   0 (N-ATR A1 terminus) 2   3 R RYY A1 3  17 M QSD A1 4  18 Q SDL A1 5  22 GELP A1 6  24 L PVD A1 7  26 V DAR A1 8  28 A RFP A1 9  32 P RVP A1 10 38 F PFN A1 11  40 F NTS A1 12  41 N TSV A1 13  60 N IAK A1 14  61 IAKP A1 15  65 R PPW A1 16  81 Y DTV A1 17 111 G AEY A1 18 116 D QTS A119 119 S QRE A1 20 120 Q REK A1 21 128 V FPG A1 22 129 F PGG A1 23 130 PGGS A1 24 182 G SLA A1 25 185 A KEK A1 26 188 K TQT A1 27 205 G KSW A128 210 S ETK A1 29 211 E TKN A1 30 216 L MQD A1 31 220 R DAA A1 32 222 AASA A1 33 223 A SAR A1 34 224 S ARA A1 35 230 K MHT A1 36 243 P GLI A137 244 G LIG A1 38 250 R KSV A1 39 318 D GME A1 40 333 P QLR A1 42 334 QLRM A1 43 336 R MKN a1 44 339 N NEE a1 45 345 D YDD a1 46 357 V VRF a147 367 S FIQ a1 48 370 S RPY a1 49 375 A KKH A2 50 376 K KHP A2 51 378 HPKT A2 52 399 V LAP A2 53 403 D DRS A2 54 405 R SYK A2 55 409 S QYL A256 416 P QRI A2 57 434 E TFK A2 58 438 T REA A2 59 441 A IQH A2 60 442 IQHE A2 61 463 I JFK A2 62 487 Y SRR A2 63 490 R LPK A2 64 492 P KGV A265 493 K GVK A2 66 494 G VKH A2 67 500 D FPI A2 68 506 G EIF A2 69 518 EDGP A2 70 556 K ESV A2 71 565 Q IMS A2 72 566 I MSD A2 73 598 P AGV A274 599 A GVQ A2 75 603 L EDP A2 76 616 S ING A2 77 686 G LWI A2 78 713 KNTG A2 79 719 Y EDS A2 80 730 L LSK A2 81 733 K NNA A2 82 745 N PPV** B83 1640 P PVL B 84 1652 R TTL B 85 1656 Q SDQ A3 86 1685 N QSP A3 871711 M SSS A3 88 1713 S SPH A3 89 1720 N RAQ A3 90 1724 S GSV A3 91 1725G SVP A3 92 1726 S VPQ A3 93 1741 G SFT A3 94 1744 T QPL A3 95 1749 RGEL A3 96 1773 V TFR A3 97 1792 Y EED A3 98 1793 E EDQ A3 99 1796 Q RQGA3 100 1798 Q GAE A3 101 1799 G AEP A3 102 1802 P RKN A3 103 1803 R KNFA3 104 1807 V KPN A3 105 1808 K PNE A3 106 1827 K DEF A3 107 1844 E KDVA3 108 1861 N TLN A3 109 1863 L NPA A3 110 1896 E RNC A3 111 1900 R APCA3 112 1904 N IQM A3 113 1905 I QME A3 114 1910 P TFK A3 115 1920 A INGA3 116 1937 D QRI A3 117 1981 G VFE A3 118 2019 N KCQ A3 119 2020 K CQTC1 120 2044 G QWA C1 121 2068 F SWI C1 122 2073 V DLL C1 123 2090 R QKFC1 124 2092 K FSS C1 125 2093 F SSL C1 126 2111 K WQT C1 127 2115 Y RGNC1 128 2120 T GTL C1 129 2125 V FFG C1 130 2171 L NSC C1 131 2173 S CSMC2 132 2188 A QIT C2 133 2223 V NNP C2 134 2224 N NPK C2 135 2227 K EWLC2 136 2268 G HQW C2 137 2277 N GKV C2 138 2278 G KVK C2 139 2290 F TPVC2 140 2332 Y C terminus CT of FVIII *Indicates an insertion point forXTEN based on the amino acid number of mature full-length human FVIII,wherein the insertion could be either on the N- or C-terminal side ofthe indicated amino acid.

In some embodiments, one or more XTEN sequences are inserted withinabout six amino acids up or down from amino acids 32, 220, 224, 336,339, 399, 416, 603, 1656, 1711, 1725, 1905, or 1910, corresponding toSEQ ID NO: 4 or any combinations thereof.

TABLE 8 Exemplary XTEN Insertion Ranges Distance XTEN FVIII BDD fromInsertion Insertion Downstream FVIII insertion No. Point ResidueSequence Domain residue*  9  32 P RVP A1 −3, +6  31  220 R DAA A1 —  34 224 S ARA A1 +5  43  336 R MKN a1 −1, +6  44  339 N NEE a1 −4, +5  52 399 V LAP A2 −6, +3  56  416 P QRI A2 +6  75  603 L EDP A2 _6 +6  851656 Q SDQ B −3, +6  87 1711 M SSS A3 −6, +1  91 1725 G SVP A3 +6 1131905 I QME A3 +6 114 1910 P TFK A3 −5, +6 *Distance from insertionresidue refers to the relative number of amino acids away from theN-terminus (negative numbers) or C-terminus (positive numbers) of thedesignated insertion residue (residue “0”) where an insertion may bemade. The designation “−x” refers to an insertion site which is x aminoacids away on the N-terminal side of the designated insertion residue.Similarly, the designation “+x” refers to an insertion site which is xamino acids away on the C-terminal side of the designated insertionresidue. For example, “−1, +2” indicates that the insertion is made atthe N-terminus or C-terminus of amino acid residues denoted −1, 0, +1 or+2.

In other embodiments, one or more XTEN sequences are insertedimmediately down stream of one or more amino acids corresponding to thefull-length mature human FVIII selected from the group consisting of oneor more insertion sites in Table 9.

TABLE 9 Exemplary XTEN Insertion Sites or Ranges XTEN Insertion FirstInsertion FVIII No. Point Range* Residue Domain  3  18-32 Q A1  8  40 FA1 18  211-224 E A1 27  336-403 R A1, A2 43  599 A A2 47  745-1640 N B50 1656-1728 Q B, a3, A3 57 1796-1804 R A3 65 1900-1912 R A3 812171-2332 L C1, C2 *indicates range of insertion sites numbered relativeto the amino acid number of mature human FVIII

In yet other embodiments, one or more XTENs are inserted in the B domainof FVIII. In one example, an XTEN is inserted between amino acids 740and 1640 corresponding to SEQ ID NO: 4, wherein the FVIII sequencebetween amino acids 740 and 1640 is optionally not present. In anotherexample, an XTEN is inserted between amino acids 741 and 1690corresponding to SEQ ID NO: 4, wherein the FVIII sequence between aminoacids 740 and 1690 is optionally not present. In other examples, an XTENis inserted between amino acids 741 and 1648 corresponding to SEQ ID NO:4, wherein the FVIII sequence between amino acids 741 and 1648 isoptionally not present. In yet other examples, an XTEN is insertedbetween amino acids 743 and 1638 corresponding to SEQ ID NO: 4, whereinthe FVIII sequence between amino acids 743 and 1638 is optionally notpresent. In still other examples, an XTEN is inserted between aminoacids 745 and 1656 corresponding to SEQ ID NO: 4, wherein the FVIIIsequence between amino acids 745 and 1656 is optionally not present. Insome examples, an XTEN is inserted between amino acids 745 and 1657corresponding to SEQ ID NO: 4, wherein the FVIII sequence between aminoacids 745 and 1657 is optionally not present. In certain examples, anXTEN is inserted between amino acids 745 and 1667 corresponding to SEQID NO: 4, wherein the FVIII sequence between amino acids 745 and 1667 isoptionally not present. In still other examples, an XTEN is insertedbetween amino acids 745 and 1686 corresponding to SEQ ID NO: 4, whereinthe FVIII sequence between amino acids 745 and 1686 is optionally notpresent. In some other examples, an XTEN is inserted between amino acids747 and 1642 corresponding to SEQ ID NO: 4, wherein the FVIII sequencebetween amino acids 747 and 1642 is optionally not present. In stillother examples, an XTEN is inserted between amino acids 751 and 1667corresponding to SEQ ID NO: 4, wherein the FVIII sequence between aminoacids 751 and 1667 is optionally not present.

In some embodiments, one or more XTENs are inserted in one or more aminoacids immediately downstream of an amino acid of an insertion siteselected from the group consisting of the amino acid residues in Table10.

TABLE 10 FVIII XTEN insertion sites and construct designations Up- Down-stream stream Construct Residue Residue Upstream Downstream NumberDomain No.* No.* Sequence Sequence F8X-1 A1   3   4 ATR RYY F8X-2 A1  18 19 YMQ SDL F8X-3 A1  22  23 DLG ELP F8X-4 A1  26  27 LPV DAR F8X-5 A1 40  41 FPF NTS F8X-6 A1  60  61 LFN IAK F8X-7 A1  116  117 YDD QTSF8X-8 A1  130  131 VFP GGS F8X-9 A1  188  189 KEK TQT F8X-10 A1  216 217 NSL MQD F8X-11 A1  230  231 WPK MHT F8X-12 A1  333  334 EEP QLRF8X-13 A2  375  376 SVA KKH F8X-14 A2  403  404 APD DRS F8X-15 A2  442 443 EAI QHE F8X-16 A2  490  491 RRL PKG F8X-17 A2  518  519 TVE DGPF8X-18 A2  599  600 NPA GVQ F8X-19 A2  713  714 CDK NTG F8X-20 BD  745 746 SQN PPV F8X-21 BD  745  746 SQN PPV F8X-22 BD**  745  746 SQN PPVF8X-23 A3 1720 1721 APT KDE F8X-24 A3 1796 1797 EDQ RQG F8X-25 A3 18021803 AEP RKN F8X-26 A3 1827 1828 PTK DEF F8X-27 A3 1861 1862 HTN TLNF8X-28 A3 1896 1897 NME RNC F8X-29 A3 1900 1901 NCR APC F8X-30 A3 19041905 PCN TQM F8X-31 A3 1937 1938 AQD QRI F8X-32 C1 2019 2020 YSN KCQF8X-33 C1 2068 2069 EPF SWI F8X-34 C1 2111 2112 GKK WQT F8X-35 C1 21202121 NST GTL F8X-36 C2 2171 2172 CDL NSC F8X-37 C2 2188 2189 SDA QITF8X-38 C2 2227 2228 NPK EWL F8X-39 C2 2277 2278 FQN GKV F8X-40 CT 2332NA DLY NA F8X-41 CT 2332 NA DLY NA F8X-42 A1   3   4 ATR ATR pSD0001 A2 403  404 pSD0002 A2  599  600 pSD0021 N-term   0   1 pSD0022 A1  32  33pSD0023 A1  65  66 pSD0024 A1  81  82 pSD0025 A1  119  120 pSD0026 A1 211  212 pSD0027 A1  220  221 pSD0028 A1  224  225 pSD0029 A1  336  337pSD0030 A1  339  340 pSD0031 A2  378  379 pSD0032 A2  399  400 pSD0033A2  409  410 pSD0034 A2  416  417 pSD0035 A2  487  488 pSD0036 A2  494 495 pSD0037 A2  500  501 pSD0038 A2  603  604 pSD0039 A3 1656 1657pSD0040 A3 1711 1712 pSD0041 A3 1725 1726 pSD0042 A3 1749 1750 pSD0043A3 1905 1906 pSD0044 A3 1910 1911 pDS0062 A3 1900 1901 * Indicates theamino acid number of the mature FVIII protein

In one embodiment, the one or more XTEN insertion sites are locatedwithin one or more surface-exposed, flexible loop structure of the FVIIIprotein (e.g., a permissive loop). For example, at least one XTENsequence can be inserted in each FVIII “A” domain comprising at leasttwo “permissive loops” into which at least one XTEN polypeptide can beinserted without eliminating procoagulant activity of the recombinantprotein, or the ability of the recombinant proteins to be expressed invivo or in vitro in a host cell. The permissive loops are regions thatallow insertion of at least one XTEN sequence with, among otherattributes, high surface or solvent exposure and high conformationalflexibility. The A1 domain comprises a permissive loop-1 (A1-1) regionand a permissive loop-2 (A1-2) region, the A2 domain comprises apermissive loop-1 (A2-1) region and a permissive loop-2 (A2-2) region,the A3 domain comprises a permissive loop-1 (A3-1) region and apermissive loop-2 (A3-2) region.

In one aspect, a first permissive loop in the FVIII A1 domain (A1-1) islocated between beta strand 1 and beta strand 2, and a second permissiveloop in the FVIII A2 domain (A1-2) is located between beta strand 11 andbeta strand 12. A first permissive loop in the FVIII A2 domain (A2-1) islocated between beta strand 22 and beta strand 23, and a secondpermissive loop in the FVIII A2 domain (A2-2) is located between betastrand 32 and beta strand 33. A first permissive loop in the FVIII A3domain (A3-1) is located between beta strand 38 and beta strand 39, anda second permissive loop in the FVIII A3 (A3-2) is located between betastrand 45 and beta strand 46. In certain aspects, the surface-exposed,flexible loop structure comprising A1-1 corresponds to a region innative mature human FVIII from about amino acid 15 to about amino acid45 of SEQ ID NO: 4, e.g., from about amino acid 18 to about amino acid41 of SEQ ID NO: 4. In other aspects, the surface-exposed, flexible loopstructure comprising A1-2 corresponds to a region in native mature humanFVIII from about amino acid 201 to about amino acid 232 of SEQ ID NO: 4,e.g., from about amino acid 218 to about amino acid 229 of SEQ ID NO: 4.In yet other aspects, the surface-exposed, flexible loop structurecomprising A2-1 corresponds to a region in native mature human FVIIIfrom about amino acid 395 to about amino acid 421 of SEQ ID NO: 4, e.g.from about amino acid 397 to about amino acid 418 of SEQ ID NO: 4. Instill other embodiments, the surface-exposed, flexible loop structurecomprising A2-2 corresponds to a region in native mature human FVIIIfrom about amino acid 577 to about amino acid 635 of SEQ ID NO: 4, e.g.,from about amino acid 595 to about amino acid 607 of SEQ ID NO: 4. Incertain aspects the surface-exposed, flexible loop structure comprisingA3-1 corresponds to a region in native mature human FVIII from aboutamino acid 1705 to about amino acid 1732 of SEQ ID NO: 4, e.g., fromabout amino acid 1711 to about amino acid 1725 of SEQ ID NO: 4. In yetother aspects, the surface-exposed, flexible loop structure comprisingA3-2 corresponds to a region in native mature human FVIII from aboutamino acid 1884 to about amino acid 1917 of SEQ ID NO: 4, e.g., fromabout amino acid 1899 to about amino acid 1911 of SEQ ID NO: 4.

In another embodiment, the one or more amino acids in which at least oneXTEN sequence is inserted is located within a3 domain, e.g., amino acids1649 to 1689, corresponding to full-length mature FVIII polypeptide. Ina particular embodiment, an XTEN sequence is inserted between aminoacids 1656 and 1657 of SEQ ID NO: 4 (full-length mature FVIII). In aspecific embodiment, a FVIII protein comprising an XTEN sequenceinserted immediately downstream of amino acid 1656 corresponding to SEQID NO: 4 further comprises a deletion from amino acid 745 to amino acid1656 corresponding to SEQ ID NO: 4.

In some embodiments, the one or more insertion sites for one or moreXTEN insertions are immediately downstream of one or more amino acidsselected from the group consisting of:

  (1) amino acid 3,   (2) amino acid 18, (3) amino acid 22, (4) aminoacid 26, (5) amino acid 32, (6) amino acid 40, (7) amino acid 60, (8)amino acid 65, (9) amino acid 81, (10) amino acid 116, (11) amino acid119, (12) amino acid 130, (13) amino acid 188, (14) amino acid 211, (15)amino acid 216, (16) amino acid 220, (17) amino acid 224, (18) aminoacid 230, (19) amino acid 333, (20) amino acid 336, (21) amino acid 339,(22) amino acid 375, (23) amino acid 399, (24) amino acid 403, (25)amino acid 409, (26) amino acid 416, (26) amino acid 442, (28) aminoacid 487, (29) amino acid 490, (30) amino acid 494, (31) amino acid 500,(32) amino acid 518, (33) amino acid 599, (34) amino acid 603, (35)amino acid 713, (36) amino acid 745, (37) amino acid 1656, (38) aminoacid 1711, (39) amino acid 1720, (40) amino acid 1725, (41) amino acid1749, (42) amino acid 1796, (43) amino acid 1802, (44) amino acid 1827,(45) amino acid 1861, (46) amino acid 1896, (47) amino acid 1900, (48)amino acid 1904, (49) amino acid 1905, (50) amino acid 1910, (51) aminoacid 1937, (52) amino acid 2019, (53) amino acid 2068, (54) amino acid2111, (55) amino acid 2120, (56) amino acid 2171, (57) amino acid 2188,(58) amino acid 2227, (59) amino acid 2277, and (60) two or morecombinations thereof.

In one embodiment, a FVIII protein useful for the invention comprisestwo XTEN sequences, a first XTEN sequence inserted into a first XTENinsertion site and a second XTEN inserted into a second XTEN insertionsite. Non-limiting examples of the first XTEN insertion site and thesecond XTEN insertion site are listed in Table 11.

TABLE 11 Exemplary Insertion Sites for Two XTENs Insertion 1 Insertion 2Insertion Site Domain Insertion Site Domain  745 B 2332 CT  26 A1  403A2  40 A1  403 A2  18 A1  403 A2  26 A1  599 A2  40 A1  599 A2  18 A1 599 A2 1720 A3 1900 A3 1725 A3 1900 A3 1711 A3 1905 A3 1720 A3 1905 A31725 A3 1905 A3 1656 A3  26 A1 1656 A3  18 A1 1656 A3  40 A1 1656 A3 399 A2 1656 A3  403 A2 1656 A3 1725 A3 1656 A3 1720 A3 1900 A3  18 A11900 A3  26 A1 1900 A3  40 A1 1905 A3  18 A1 1905 A3  40 A1 1905 A3  26A1 1910 A3  26 A1  18 A1  399 A2  26 A1  399 A2  40 A1  399 A2  18 A1 403 A2 1656 A3 1900 A3 1656 A3 1905 A3 1711 A3  40 A1 1711 A3  26 A11720 A3  26 A1 1720 A3  40 A1 1720 A3  18 A1 1725 A3  26 A1 1725 A3  40A1 1725 A3  18 A1 1720 A3  403 A2 1720 A3  399 A2 1711 A3  403 A2 1720A3  403 A2 1725 A3  403 A2 1725 A3  399 A2 1711 A3  403 A2 1900 A3  399A2 1900 A3  403 A2 1905 A3  403 A2 1905 A3  399 A2 1910 A3  403 A2

The two XTENs inserted or linked to the FVIII protein can be identicalor different. In some embodiments, a FVIII protein useful for theinvention comprises two XTEN sequences inserted in the FVIII protein, afirst XTEN sequence inserted immediately downstream of amino acid 745corresponding to SEQ ID NO: 4, and a second XTEN sequence insertedimmediately downstream of amino acid 2332 corresponding to SEQ ID NO: 4(the C-terminus). In other embodiments, the first XTEN sequence isinserted immediately downstream of amino acid 18, 26, 40, 1656, or 1720corresponding to SEQ ID NO: 4, and a second XTEN sequence insertedimmediately downstream of amino acid 403 corresponding to SEQ ID NO: 4.In yet other embodiments, the first XTEN sequence is insertedimmediately downstream of amino acid 18, 26, or 40 corresponding to SEQID NO: 4, and a second XTEN sequence inserted immediately downstream ofamino acid 599 corresponding to SEQ ID NO: 4. In still otherembodiments, the first XTEN sequence is inserted immediately downstreamof amino acid 1656 corresponding to SEQ ID NO: 4, and a second XTENsequence inserted immediately downstream of amino acid 18, 26, 40, 399,403, 1725, 1720, 1900, 1905, or 2332 corresponding to SEQ ID NO: 4. Incertain embodiments, the first XTEN sequence is inserted immediatelydownstream of amino acid 1900 corresponding to SEQ ID NO: 4, and asecond XTEN sequence inserted immediately downstream of amino acid 18,26, or 40 corresponding to SEQ ID NO: 4. In some embodiments, the firstXTEN sequence is inserted immediately downstream of amino acid 18, 26,or 40 corresponding to SEQ ID NO: 4, and a second XTEN sequence insertedimmediately downstream of amino acid 399 corresponding to SEQ ID NO: 4.In other embodiments, the first XTEN sequence is inserted immediatelydownstream of amino acid 1720 corresponding to SEQ ID NO: 4, and asecond XTEN sequence inserted immediately downstream of amino acid 18,26, or 40 corresponding to SEQ ID NO: 4. In still other embodiments, thefirst XTEN sequence is inserted immediately downstream of amino acid1720 corresponding to SEQ ID NO: 4, and a second XTEN sequence insertedimmediately downstream of amino acid 18 corresponding to SEQ ID NO: 4.In a particular embodiment, the FVIII protein comprising two XTENsequences, a first XTEN sequence inserted immediately downstream ofamino acid 745 corresponding to SEQ ID NO: 4 and a second XTEN sequenceinserted immediately downstream of amino acid 2332 corresponding to SEQID NO: 4, wherein the FVIII protein further has a deletion from aminoacid 745 corresponding to SEQ ID NO: 4 to amino acid 1685 correspondingto SEQ ID NO: 4, a mutation or substitution at amino acid 1680corresponding to SEQ ID NO: 4, e.g., Y1680F, a mutation or substitutionat amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A, or atleast two mutations or substitutions at amino acid 1648 corresponding toSEQ ID NO: 4, e.g., R1648A, and amino acid 1680 corresponding to SEQ IDNO: 4, e.g., Y1680F. In a specific embodiment, the FVIII proteincomprises two XTEN sequences, a first XTEN inserted immediatelydownstream of amino acid 1656 corresponding to SEQ ID NO: 4 and a secondXTEN sequence inserted immediately downstream of amino acid 2332 of SEQID NO: 4, wherein the FVIII protein further has a deletion from aminoacid 745 to amino acid 1656 corresponding to SEQ ID NO: 4.

In certain embodiments, a FVIII protein comprises three XTEN sequences,a first XTEN sequence inserted into a first XTEN insertion site, asecond XTEN sequence inserted into a second XTEN sequence, and a thirdXTEN sequence inserted into a third XTEN insertion site. The first,second, or third XTEN sequences can be identical or different. Thefirst, second, and third insertion sites can be selected from the groupof any one of the insertion sites disclosed herein. In some embodiments,the FVIII protein comprising three XTEN sequences can further comprise amutation or substitution, e.g., amino acid 1648 corresponding to SEQ IDNO: 4, e.g., R1648A. For example, non-limiting examples of the first,second, and third XTEN insertion sites are listed in Table 12.

TABLE 12 Exemplary Insertion Sites for Three XTENs Insertion 1 Insertion2 Insertion 3 Insertion Insertion Insertion Site Domain Site Domain SiteDomain  26 A1  403 A2 1656 A3  26 A1  403 A2 1720 A3  26 A1  403 A2 1900A3  26 A1 1656 A3 1720 A3  26 A1 1656 A3 1900 A3  26 A1 1720 A3 1900 A3 403 A2 1656 A3 1720 A3  403 A2 1656 A3 1900 A3  403 A2 1720 A3 1900 A31656 A3 1720 A3 1900 A3  745 B 1900 2332 CT  18 A1  745 B 2332 CT  26 A1 745 B 2332 CT  40 A1  745 B 2332 CT  18 A1  745 B 2332 CT  40 A1  745 B2332 CT  403 A2  745 B 2332 CT  399 A2  745 B 2332 CT 1725 A3  745 B2332 CT 1720 A3  745 B 2332 CT 1711 A3  745 B 2332 CT 1900 A3  745 B2332 CT 1905 A3  745 B 2332 CT 1910 A3  745 B 2332 CT

In some embodiments, a FVIII protein comprises three XTEN sequences, afirst XTEN sequence inserted immediately downstream of amino acid 26corresponding to SEQ ID NO: 4, a second XTEN sequence inserteddownstream of amino acid 403 corresponding to SEQ ID NO: 4, and a thirdXTEN sequence inserted downstream of amino acid 1656, 1720, or 1900corresponding to SEQ ID NO: 4. In other embodiments, the first XTENsequence is inserted immediately downstream of amino acid 26corresponding to SEQ ID NO: 4, a second XTEN sequence is inserteddownstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a thirdXTEN sequence is inserted downstream of amino acid 1720 or 1900corresponding to SEQ ID NO: 4. In yet other embodiments, the first XTENsequence is inserted immediately downstream of amino acid 26corresponding to SEQ ID NO: 4, a second XTEN sequence is inserteddownstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a thirdXTEN sequence is inserted downstream of amino acid 1900 corresponding toSEQ ID NO: 4. In still other embodiments, the first XTEN sequence isinserted immediately downstream of amino acid 403 corresponding to SEQID NO: 4, a second XTEN sequence is inserted downstream of amino acid1656 corresponding to SEQ ID NO: 4, and a third XTEN sequence isinserted downstream of amino acid 1720 or 1900 corresponding to SEQ IDNO: 4. In other embodiments, the first XTEN sequence is insertedimmediately downstream of amino acid 403 or 1656 corresponding to SEQ IDNO: 4, a second XTEN sequence is inserted downstream of amino acid 1720corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserteddownstream of amino acid 1900 corresponding to SEQ ID NO: 4. In otherembodiments, the first XTEN sequence is inserted immediately downstreamof amino acid 18, 26, 40, 399, 403, 1711, 1720, 1725, 1900, 1905, or1910 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserteddownstream of amino acid 745 corresponding to SEQ ID NO: 4, and a thirdXTEN sequence is inserted downstream of amino acid 2332 corresponding toSEQ ID NO: 4.

In other embodiments, a FVIII protein in the invention comprises fourXTEN sequences, a first XTEN sequence inserted into a first insertionsite, a second XTEN sequence inserted into a second insertion site, athird XTEN sequence inserted into a third insertion site, and a fourthXTEN sequence inserted into a fourth insertion site. The first, second,third, and fourth XTEN sequences can be identical, different, orcombinations thereof. In some embodiments, the FVIII protein comprisingfour XTEN sequences can further comprise a mutation or substitution,e.g., amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A.Non-limiting examples of the first, second, third, and fourth XTENinsertion sites are listed in Table 13.

TABLE 13 Exemplary Insertion Sites for Four XTENs Insertion 1 Insertion2 Insertion 3 Insertion 4 Insertion Do- Insertion Do- Insertion Do-Insertion Do- Site main Site main Site main Site main 26 A1 403 A2 1656a3 1720 A3 26 A1 403 A2 1656 a3 1900 A3 26 A1 403 A2 1720 A3 1900 A3 26A1 1656 a3 1720 A3 1900 A3 403 A2 1656 a3 1720 A3 1900 A3 0040 A1 0403A2 745 B 2332 CT 0040 A1 0403 A2 745 B 2332 CT 0018 A1 0409 A2 745 B2332 CT 0040 A1 0409 A2 745 B 2332 CT 0040 A1 0409 A2 745 B 2332 CT 0018A1 0409 A2 745 B 2332 CT 0040 A1 1720 A3 745 B 2332 CT 0026 A1 1720 A3745 B 2332 CT 0018 A1 1720 A3 745 B 2332 CT 0018 A1 1720 A3 745 B 2332CT 0018 A1 1720 A3 745 B 2332 CT 0026 A1 1720 A3 745 B 2332 CT 0018 A11720 A3 745 B 2332 CT 0018 A1 1900 A3 745 B 2332 CT 0018 A1 1900 A3 745B 2332 CT 0026 A1 1900 A3 745 B 2332 CT 0040 A1 1900 A3 745 B 2332 CT0040 A1 1905 A3 745 B 2332 CT 0018 A1 1905 A3 745 B 2332 CT 0040 A1 1905A3 745 B 2332 CT 0026 A1 1905 A3 745 B 2332 CT 0018 A1 1905 A3 745 B2332 CT 0018 A1 1905 A3 745 B 2332 CT 0018 A1 1910 A3 745 B 2332 CT 0018A1 1910 A3 745 B 2332 CT 0040 A1 1910 A3 745 B 2332 CT 0026 A1 1910 A3745 B 2332 CT 0018 A1 1910 A3 745 B 2332 CT 0026 A1 1910 A3 745 B 2332CT 0040 A1 1910 A3 745 B 2332 CT 0018 A1 1910 A3 745 B 2332 CT 0409 A21720 A3 745 B 2332 CT 0403 A2 1720 A3 745 B 2332 CT 0409 A2 1720 A3 745B 2332 CT 0403 A2 1720 A3 745 B 2332 CT 0403 A2 1720 A3 745 B 2332 CT0403 A2 1900 A3 745 B 2332 CT 0403 A2 1900 A3 745 B 2332 CT 0409 A2 1900A3 745 B 2332 CT 0403 A2 1900 A3 745 B 2332 CT 0403 A2 1900 A3 745 B2332 CT 0409 A2 1900 A3 745 B 2332 CT 0409 A2 1905 A3 745 B 2332 CT 0403A2 1905 A3 745 B 2332 CT 0403 A2 1905 A3 745 B 2332 CT 0403 A2 1905 A3745 B 2332 CT 0409 A2 1905 A3 745 B 2332 CT 0403 A2 1905 A3 745 B 2332CT 0409 A2 1910 A3 745 B 2332 CT 0403 A2 1910 A3 745 B 2332 CT 0403 A21910 A3 745 B 2332 CT 0403 A2 1910 A3 745 B 2332 CT 0403 A2 1910 A3 745B 2332 CT 1720 A3 1900 A3 745 B 2332 CT 1720 A3 1905 A3 745 B 2332 CT1720 A3 1910 A3 745 B 2332 CT 1720 A3 1910 A3 745 B 2332 CT 0403 A2 1656a3 1720 A3 2332 CT 0403 A2 1656 a3 1900 A3 2332 CT 0403 A2 1720 A3 1900A3 2332 CT 1656 a3 1720 A3 1900 A3 2332 CT 0018 A1 0403 A2 1656 a3 2332CT 0018 A1 0403 A2 1720 A3 2332 CT 0018 A1 0403 A2 1900 A3 2332 CT 0018A1 1656 a3 1720 A3 2332 CT 0018 A1 1656 a3 1900 A3 2332 CT 0018 A1 1720A3 1900 A3 2332 CT 0018 A1 0403 A2 0745 B 2332 CT 0018 A1 0745 B 1720 A32332 CT 0018 A1 0745 B 1900 A3 2332 CT 0403 A2 0745 B 1720 A3 2332 CT0403 A2 0745 B 1900 A3 2332 CT 0745 B 1720 A3 1900 A3 2332 CT 0188 A11900 A3 0745 B 2332 CT 0599 1900 A3 0745 B 2332 CT 2068 1900 A3 0745 B2332 CT 2171 1900 A3 0745 B 2332 CT 2227 1900 A3 0745 B 2332 CT 22771900 A3 0745 B 2332 CT

In some embodiments, a FVIII protein comprises five XTEN sequences, afirst XTEN sequence inserted into a first insertion site, a second XTENsequence inserted into a second insertion site, a third XTEN sequenceinserted into a third XTEN insertion site, a fourth XTEN sequenceinserted into a fourth XTEN insertion site, and a fifth XTEN sequenceinserted into a fifth XTEN insertion site. The first, second, third,fourth, of fifth XTEN sequences can be identical, different, orcombinations thereof. Non-limiting examples of the first, second, third,fourth, and fifth insertion sites are listed in Table 14.

TABLE 14 Exemplary Insertion Sites for Five XTENs XTEN XTEN XTEN XTENXTEN Insertion 1 insertion 2 Insertion 3 Insertion 4 Insertion 5 04031656 1720 1900 2332 0018 0403 1656 1720 2332 0018 0403 1656 1900 23320018 0403 1720 1900 2332 0018 1656 1720 1900 2332 0018 0403 0745 17202332 0018 0403 0745 1900 2332 0018 0745 1720 1900 2332 0403 0745 17201900 2332

In certain embodiments, a FVIII protein comprises six XTEN sequences, afirst XTEN sequence inserted into a first XTEN insertion site, a secondXTEN sequence inserted into a second XTEN insertion site, a third XTENsequence inserted into a third XTEN insertion site, a fourth XTENsequence inserted into a fourth XTEN insertion site, a fifth XTENsequence inserted into a fifth XTEN insertion site, and a sixth XTENsequence inserted into a sixth XTEN insertion site. The first, second,third, fourth, fifth, or sixth XTEN sequences can be identical,different, or combinations thereof. Examples of the six XTEN insertionsites include, but are not limited to the insertion sites listed inTable 15.

TABLE 15 Exemplary XTEN Insertion Sites for Six XTENs XTEN XTEN XTENXTEN XTEN XTEN Inser- inser- Inser- Inser- Inser- Inser- tion 1 tion 2tion 3 tion 4 tion 5 tion 6 0018 0403 1656 1720 1900 2332 0018 0403 07451720 1900 2332

In a particular example, a first XTEN is inserted between amino acids 26and 27 corresponding to SEQ ID NO: 4, and a second XTEN is insertedbetween amino acids 1720 and 1721 corresponding to SEQ ID NO: 4(full-length mature FVIII). In another example, a first XTEN is insertedbetween amino acids 403 and 404 corresponding to SEQ ID NO: 4, and asecond XTEN is inserted between amino acids 1720 and 1721 correspondingto SEQ ID NO: 4. In some examples, a first XTEN is inserted betweenamino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a secondXTEN is inserted between amino acids 1720 and 1721 corresponding to SEQID NO: 4. In other examples, a first XTEN is inserted between aminoacids 26 and 27 corresponding to SEQ ID NO: 4, a second XTEN is insertedbetween amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and athird XTEN is inserted between amino acids 1720 and 1721 correspondingto SEQ ID NO: 4. In yet other embodiments, a first XTEN is insertedbetween amino acids 403 and 404 corresponding to SEQ ID NO: 4, a secondXTEN is inserted between amino acids 1656 and 1657 corresponding to SEQID NO: 4, and a third XTEN is inserted between amino acids 1720 and 1721corresponding to SEQ ID NO: 4. In still other embodiments, a first XTENis inserted between amino acids 403 and 404 corresponding to SEQ ID NO:4, a second XTEN is inserted between amino acids 1656 and 1657corresponding to SEQ ID NO: 4, and a third XTEN is inserted betweenamino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In certainembodiments, a first XTEN is inserted between amino acids 26 and 27corresponding to SEQ ID NO: 4, a second XTEN is inserted between aminoacids 1720 and 1721 corresponding to SEQ ID NO: 4, and a third XTEN isinserted between amino acids 1900 and 1901 corresponding to SEQ ID NO:4. In some embodiments, a first XTEN is inserted between amino acids 26and 27 corresponding to SEQ ID NO: 4, a second XTEN is inserted betweenamino acids 1656 and 1657 corresponding to SEQ ID NO: 4, a third XTEN isinserted between amino acids 1720 and 1721 corresponding to SEQ ID NO:4, and a fourth XTEN is inserted between 1900 and 1901 corresponding toSEQ ID NO: 4.

In a particular embodiment, an XTEN sequence is inserted between aminoacids 745 and 746 of a full-length Factor VIII or the correspondinginsertion site of the B-domain deleted Factor VIII.

In some embodiments, a chimeric protein of the invention comprises twopolypeptide sequences, a first polypeptide sequence comprising an aminoacid sequence at least about 80%, 90%, 95%, or 100% identical to asequence selected from FVIII-161 (SEQ ID NO: 101), FVIII-169 (SEQ ID NO:103), FVIII-170 (SEQ ID NO: 102), FVIII-173 (SEQ ID NO: 104); FVIII-195(SEQ ID NO: 105); FVIII-196 (SEQ ID NO: 106), FVIII-199 (SEQ ID NO:107), FVIII-201 (SEQ ID NO: 108); FVIII-203 (SEQ ID NO: 109), FVIII-204(SEQ ID NO: 110), FVIII-205 (SEQ ID NO: 111), FVIII-266 (SEQ ID NO:112),FVIII-267 (SEQ ID NO: 113), FVIII-268 (SEQ ID NO: 114), FVIII-269 (SEQID NO: 115), FVIII-271 (SEQ ID NO: 116), or FVIII-272 (SEQ ID NO: 117)and a second polypeptide sequence comprising an amino acid sequence atleast about 80%, 90%, 95%, or 100% identical to a sequence selected fromVWF031 (SEQ ID NO: 118), VWF034 (SEQ ID NO: 119), or VWF-036 (SEQ ID NO:120).

D) Ig Constant Region or a Portion Thereof

The VWF fragment or the FVIII protein linked to an XTEN sequence in thepresent invention can further comprise an Ig constant region or aportion thereof. The Ig constant region or a portion thereof can improvepharmacokinetic or pharmacodynamic properties of the VWF fragment or theFVIII protein in combination with the XTEN sequence. In certainembodiments, the Ig constant region or a portion thereof extends ahalf-life of a molecule fused to the Ig constant region or a portionthereof.

An Ig constant region is comprised of domains denoted CH (constantheavy) domains (CH1, CH2, etc.). Depending on the isotype, (i.e. IgG,IgM, IgA, IgD, or IgE), the constant region can be comprised of three orfour CH domains. Some isotypes (e.g. IgG) constant regions also containa hinge region. See Janeway et al. 2001, Immunobiology, GarlandPublishing, N.Y., N.Y.

An Ig constant region or a portion thereof for producing the chimericprotein of the present invention may be obtained from a number ofdifferent sources. In some embodiments, an Ig constant region or aportion thereof is derived from a human Ig. It is understood, however,that the Ig constant region or a portion thereof may be derived from anIg of another mammalian species, including for example, a rodent (e.g. amouse, rat, rabbit, guinea pig) or non-human primate (e.g. chimpanzee,macaque) species. Moreover, the Ig constant region or a portion thereofmay be derived from any Ig class, including IgM, IgG, IgD, IgA, and IgE,and any Ig isotype, including IgG1, IgG2, IgG3, and IgG4. In oneembodiment, the human isotype IgG1 is used.

A variety of the Ig constant region gene sequences (e.g., human constantregion gene sequences) are available in the form of publicly accessibledeposits. Constant region domains sequence can be selected having aparticular effector function (or lacking a particular effector function)or with a particular modification to reduce immunogenicity. Manysequences of antibodies and antibody-encoding genes have been publishedand suitable Ig constant region sequences (e.g., hinge, CH2, and/or CH3sequences, or portions thereof) can be derived from these sequencesusing art recognized techniques. The genetic material obtained using anyof the foregoing methods may then be altered or synthesized to obtainpolypeptides of the present invention. It will further be appreciatedthat the scope of this invention encompasses alleles, variants andmutations of constant region DNA sequences.

The sequences of the Ig constant region or a portion thereof can becloned, e.g., using the polymerase chain reaction and primers which areselected to amplify the domain of interest. To clone a sequence of theIg constant region or a portion thereof from an antibody, mRNA can beisolated from hybridoma, spleen, or lymph cells, reverse transcribedinto DNA, and antibody genes amplified by PCR. PCR amplification methodsare described in detail in U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; 4,965,188; and in, e.g., “PCR Protocols: A Guide to Methodsand Applications” Innis et al. eds., Academic Press, San Diego, Calif.(1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol.217:270). PCR may be initiated by consensus constant region primers orby more specific primers based on the published heavy and light chainDNA and amino acid sequences. As discussed above, PCR also may be usedto isolate DNA clones encoding the antibody light and heavy chains. Inthis case the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes. Numerous primersets suitable for amplification of antibody genes are known in the art(e.g., 5′ primers based on the N-terminal sequence of purifiedantibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapidamplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods173:33); antibody leader sequences (Larrick et al. 1989 Biochem.Biophys. Res. Commun. 160:1250). The cloning of antibody sequences isfurther described in Newman et al., U.S. Pat. No. 5,658,570, filed Jan.25, 1995, which is incorporated by reference herein.

An Ig constant region used herein can include all domains and the hingeregion or portions thereof. In one embodiment, the Ig constant region ora portion thereof comprises CH2 domain, CH3 domain, and a hinge region,i.e., an Fc region or an FcRn binding partner.

As used herein, the term “Fc region” is defined as the portion of apolypeptide which corresponds to the Fc region of native Ig, i.e., asformed by the dimeric association of the respective Fc domains of itstwo heavy chains. A native Fc region forms a homodimer with another Fcregion. In contrast, the term “genetically-fused Fc region” or“single-chain Fc region” (scFc region), as used herein, refers to asynthetic dimeric Fc region comprised of Fc domains genetically linkedwithin a single polypeptide chain (i.e., encoded in a single contiguousgenetic sequence).

In one embodiment, the “Fc region” refers to the portion of a single Igheavy chain beginning in the hinge region just upstream of the papaincleavage site (i.e. residue 216 in IgG, taking the first residue ofheavy chain constant region to be 114) and ending at the C-terminus ofthe antibody. Accordingly, a complete Fc domain comprises at least ahinge domain, a CH2 domain, and a CH3 domain.

The Fc region of an Ig constant region, depending on the Ig isotype caninclude the CH2, CH3, and CH4 domains, as well as the hinge region.Chimeric proteins comprising an Fc region of an Ig bestow severaldesirable properties on a chimeric protein including increasedstability, increased serum half-life (see Capon et al., 1989, Nature337:525) as well as binding to Fc receptors such as the neonatal Fcreceptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726, 6,030,613; WO03/077834; US2003-0235536A1), which are incorporated herein by referencein their entireties.

An Ig constant region or a portion thereof can be an FcRn bindingpartner. FcRn is active in adult epithelial tissues and expressed in thelumen of the intestines, pulmonary airways, nasal surfaces, vaginalsurfaces, colon and rectal surfaces (U.S. Pat. No. 6,485,726). An FcRnbinding partner is a portion of an Ig that binds to FcRn.

The FcRn receptor has been isolated from several mammalian speciesincluding humans. The sequences of the human FcRn, monkey FcRn, ratFcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med.180:2377). The FcRn receptor binds IgG (but not other Ig classes such asIgA, IgM, IgD, and IgE) at relatively low pH, actively transports theIgG transcellularly in a luminal to serosal direction, and then releasesthe IgG at relatively higher pH found in the interstitial fluids. It isexpressed in adult epithelial tissue (U.S. Pat. Nos. 6,485,726,6,030,613, 6,086,875; WO 03/077834; US2003-0235536A1) including lung andintestinal epithelium (Israel et al. 1997, Immunology 92:69) renalproximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol.Renal Physiol. 282:F358) as well as nasal epithelium, vaginal surfaces,and biliary tree surfaces.

FcRn binding partners useful in the present invention encompassmolecules that can be specifically bound by the FcRn receptor includingwhole IgG, the Fc fragment of IgG, and other fragments that include thecomplete binding region of the FcRn receptor. The region of the Fcportion of IgG that binds to the FcRn receptor has been described basedon X-ray crystallography (Burmeister et al. 1994, Nature 372:379). Themajor contact area of the Fc with the FcRn is near the junction of theCH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavychain. The FcRn binding partners include whole IgG, the Fc fragment ofIgG, and other fragments of IgG that include the complete binding regionof FcRn. The major contact sites include amino acid residues 248,250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain andamino acid residues 385-387, 428, and 433-436 of the CH3 domain.References made to amino acid numbering of Igs or Ig fragments, orregions, are all based on Kabat et al. 1991, Sequences of Proteins ofImmunological Interest, U.S. Department of Public Health, Bethesda, Md.

Fc regions or FcRn binding partners bound to FcRn can be effectivelyshuttled across epithelial barriers by FcRn, thus providing anon-invasive means to systemically administer a desired therapeuticmolecule. Additionally, fusion proteins comprising an Fc region or anFcRn binding partner are endocytosed by cells expressing the FcRn. Butinstead of being marked for degradation, these fusion proteins arerecycled out into circulation again, thus increasing the in vivohalf-life of these proteins. In certain embodiments, the portions of Igconstant regions are an Fc region or an FcRn binding partner thattypically associates, via disulfide bonds and other non-specificinteractions, with another Fc region or another FcRn binding partner toform dimers and higher order multimers.

Two FcRn receptors can bind a single Fc molecule. Crystallographic datasuggest that each FcRn molecule binds a single polypeptide of the Fchomodimer. In one embodiment, linking the FcRn binding partner, e.g., anFc fragment of an IgG, to a biologically active molecule provides ameans of delivering the biologically active molecule orally, buccally,sublingually, rectally, vaginally, as an aerosol administered nasally orvia a pulmonary route, or via an ocular route. In another embodiment,the chimeric protein can be administered invasively, e.g.,subcutaneously, intravenously.

An FcRn binding partner region is a molecule or a portion thereof thatcan be specifically bound by the FcRn receptor with consequent activetransport by the FcRn receptor of the Fc region. Specifically boundrefers to two molecules forming a complex that is relatively stableunder physiologic conditions. Specific binding is characterized by ahigh affinity and a low to moderate capacity as distinguished fromnonspecific binding which usually has a low affinity with a moderate tohigh capacity. Typically, binding is considered specific when theaffinity constant KA is higher than 10⁶ M⁻¹, or higher than 10⁸ M⁻¹. Ifnecessary, non-specific binding can be reduced without substantiallyaffecting specific binding by varying the binding conditions. Theappropriate binding conditions such as concentration of the molecules,ionic strength of the solution, temperature, time allowed for binding,concentration of a blocking agent (e.g. serum albumin, milk casein),etc., may be optimized by a skilled artisan using routine techniques.

In certain embodiments, a chimeric protein of the invention comprisesone or more truncated Fc regions that are nonetheless sufficient toconfer Fc receptor (FcR) binding properties to the Fc region. Forexample, the portion of an Fc region that binds to FcRn (i.e., the FcRnbinding portion) comprises from about amino acids 282-438 of IgG1, EUnumbering (with the primary contact sites being amino acids 248,250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain andamino acid residues 385-387, 428, and 433-436 of the CH3 domain. Thus,an Fc region of the invention may comprise or consist of an FcRn bindingportion. FcRn binding portions may be derived from heavy chains of anyisotype, including IgG1, IgG2, IgG3 and IgG4. In one embodiment, an FcRnbinding portion from an antibody of the human isotype IgG1 is used. Inanother embodiment, an FcRn binding portion from an antibody of thehuman isotype IgG4 is used.

In another embodiment, the “Fc region” includes an amino acid sequenceof an Fc domain or derived from an Fc domain. In certain embodiments, anFc region comprises at least one of: a hinge (e.g., upper, middle,and/or lower hinge region) domain (about amino acids 216-230 of anantibody Fc region according to EU numbering), a CH2 domain (about aminoacids 231-340 of an antibody Fc region according to EU numbering), a CH3domain (about amino acids 341-438 of an antibody Fc region according toEU numbering), a CH4 domain, or a variant, portion, or fragment thereof.In other embodiments, an Fc region comprises a complete Fc domain (i.e.,a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, anFc region comprises, consists essentially of, or consists of a hingedomain (or a portion thereof) fused to a CH3 domain (or a portionthereof), a hinge domain (or a portion thereof) fused to a CH2 domain(or a portion thereof), a CH2 domain (or a portion thereof) fused to aCH3 domain (or a portion thereof), a CH2 domain (or a portion thereof)fused to both a hinge domain (or a portion thereof) and a CH3 domain (ora portion thereof). In still other embodiments, an Fc region lacks atleast a portion of a CH2 domain (e.g., all or part of a CH2 domain). Ina particular embodiment, an Fc region comprises or consists of aminoacids corresponding to EU numbers 221 to 447.

The Fc regions denoted as F, F1, or F2 herein may be obtained from anumber of different sources. In one embodiment, an Fc region of thepolypeptide is derived from a human Ig. It is understood, however, thatan Fc region may be derived from an Ig of another mammalian species,including for example, a rodent (e.g. a mouse, rat, rabbit, or guineapig) or non-human primate (e.g. chimpanzee, macaque) species. Moreover,the polypeptide of the Fc domains or portions thereof may be derivedfrom any Ig class, including IgM, IgG, IgD, IgA and IgE, and any Igisotype, including IgG1, IgG2, IgG3 and IgG4. In another embodiment, thehuman isotype IgG1 is used.

In certain embodiments, the Fc variant confers a change in at least oneeffector function imparted by an Fc region comprising said wild-type Fcdomain (e.g., an improvement or reduction in the ability of the Fcregion to bind to Fc receptors (e.g. FcγRI, FcγRII, or FcγRIII) orcomplement proteins (e.g. C1q), or to trigger antibody-dependentcytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity(CDCC)). In other embodiments, the Fc variant provides an engineeredcysteine residue.

The Fc regions of the invention may employ art-recognized Fc variantswhich are known to impart a change (e.g., an enhancement or reduction)in effector function and/or FcR or FcRn binding. Specifically, a bindingmolecule of the invention may include, for example, a change (e.g., asubstitution) at one or more of the amino acid positions disclosed inInternational PCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1,WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1,WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2,WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2,WO04/099249A2, WO05/040217A2, WO04/044859, WO05/070963A1, WO05/077981A2,WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, andWO06/085967A2; US Patent Publication Nos. US2007/0231329,US2007/0231329, US2007/0237765, US2007/0237766, US2007/0237767,US2007/0243188, US20070248603, US20070286859, US20080057056; or U.S.Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871;6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124;6,737,056; 6,821,505; 6,998,253; 7,083,784; 7,404,956, and 7,317,091,each of which is incorporated by reference herein. In one embodiment,the specific change (e.g., the specific substitution of one or moreamino acids disclosed in the art) may be made at one or more of thedisclosed amino acid positions. In another embodiment, a differentchange at one or more of the disclosed amino acid positions (e.g., thedifferent substitution of one or more amino acid position disclosed inthe art) may be made.

The Fc region or FcRn binding partner of IgG can be modified accordingto well recognized procedures such as site directed mutagenesis and thelike to yield modified IgG or Fc fragments or portions thereof that willbe bound by FcRn. Such modifications include modifications remote fromthe FcRn contact sites as well as modifications within the contact sitesthat preserve or even enhance binding to the FcRn. For example, thefollowing single amino acid residues in human IgG1 Fc (Fc γl) can besubstituted without significant loss of Fc binding affinity for FcRn:P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A,S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A,E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F,N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A,N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q,P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A,Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A,D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A,Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A,N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A,and K447A, where for example P238A represents wild type prolinesubstituted by alanine at position number 238. As an example, a specificembodiment incorporates the N297A mutation, removing a highly conservedN-glycosylation site. In addition to alanine other amino acids may besubstituted for the wild type amino acids at the positions specifiedabove. Mutations may be introduced singly into Fc giving rise to morethan one hundred Fc regions distinct from the native Fc. Additionally,combinations of two, three, or more of these individual mutations may beintroduced together, giving rise to hundreds more Fc regions. Moreover,one of the Fc region of a construct of the invention may be mutated andthe other Fc region of the construct not mutated at all, or they bothmay be mutated but with different mutations.

Certain of the above mutations may confer new functionality upon the Fcregion or FcRn binding partner. For example, one embodiment incorporatesN297A, removing a highly conserved N-glycosylation site. The effect ofthis mutation is to reduce immunogenicity, thereby enhancing circulatinghalf-life of the Fc region, and to render the Fc region incapable ofbinding to FcγR1, FcγRIIA, FcγRIIB, and FcγRIIIA, without compromisingaffinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friendet al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol.Chem. 276:6591). As a further example of new functionality arising frommutations described above affinity for FcRn may be increased beyond thatof wild type in some instances. This increased affinity may reflect anincreased “on” rate, a decreased “off” rate or both an increased “on”rate and a decreased “off” rate. Examples of mutations believed toimpart an increased affinity for FcRn include, but not limited to,T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem.276:6591).

Additionally, at least three human Fc gamma receptors appear torecognize a binding site on IgG within the lower hinge region, generallyamino acids 234-237. Therefore, another example of new functionality andpotential decreased immunogenicity may arise from mutations of thisregion, as for example by replacing amino acids 233-236 of human IgG1“ELLG” to the corresponding sequence from IgG2 “PVA” (with one aminoacid deletion). It has been shown that FcγRI, FcγRII, and FcγRIII, whichmediate various effector functions will not bind to IgG1 when suchmutations have been introduced. Ward and Ghetie 1995, TherapeuticImmunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.

In one embodiment, the Ig constant region or a portion thereof, e.g, anFc region, is a polypeptide including the sequence PKNSSMISNTP (SEQ IDNO: 52) and optionally further including a sequence selected fromHQSLGTQ (SEQ ID NO: 53), HQNLSDGK (SEQ ID NO: 54), HQNISDGK (SEQ ID NO:55), or VISSHLGQ (SEQ ID NO: 56) (U.S. Pat. No. 5,739,277).

In another embodiment, the immunoglobulin constant region or a portionthereof comprises an amino acid sequence in the hinge region or aportion thereof that forms one or more disulfide bonds with anotherimmunoglobulin constant region or a portion thereof. The disulfide bondby the immunoglobulin constant region or a portion thereof places thefirst polypeptide comprising FVIII and the second polypeptide comprisingthe VWF fragment together so that endogenous VWF does not replace theVWF fragment and does not bind to the FVIII. Therefore, the disulfidebond between the first immunoglobulin constant region or a portionthereof and a second immunoglobulin constant region or a portion thereofprevents interaction between endogenous VWF and the FVIII protein. Thisinhibition of interaction between the VWF and the FVIII protein allowsthe half-life of the FVIII protein to go beyond the two fold limit. Thehinge region or a portion thereof can further be linked to one or moredomains of CH1, CH2, CH3, a fragment thereof, and any combinationsthereof. In a particular embodiment, the immunoglobulin constant regionor a portion thereof is a hinge region and CH2.

In certain embodiments, the Ig constant region or a portion thereof ishemi-glycosylated. For example, the chimeric protein comprising two Fcregions or FcRn binding partners may contain a first, glycosylated, Fcregion (e.g., a glycosylated CH2 region) or FcRn binding partner and asecond, aglycosylated, Fc region (e.g., an aglycosylated CH2 region) orFcRn binding partner. In one embodiment, a linker may be interposedbetween the glycosylated and aglycosylated Fc regions. In anotherembodiment, the Fc region or FcRn binding partner is fully glycosylated,i.e., all of the Fc regions are glycosylated. In other embodiments, theFc region may be aglycosylated, i.e., none of the Fc moieties areglycosylated.

In certain embodiments, a chimeric protein of the invention comprises anamino acid substitution to an Ig constant region or a portion thereof(e.g., Fc variants), which alters the antigen-independent effectorfunctions of the Ig constant region, in particular the circulatinghalf-life of the protein.

Such proteins exhibit either increased or decreased binding to FcRn whencompared to proteins lacking these substitutions and, therefore, have anincreased or decreased half-life in serum, respectively. Fc variantswith improved affinity for FcRn are anticipated to have longer serumhalf-lives, and such molecules have useful applications in methods oftreating mammals where long half-life of the administered polypeptide isdesired, e.g., to treat a chronic disease or disorder (see, e.g., U.S.Pat. Nos. 7,348,004, 7,404,956, and 7,862,820). In contrast, Fc variantswith decreased FcRn binding affinity are expected to have shorterhalf-lives, and such molecules are also useful, for example, foradministration to a mammal where a shortened circulation time may beadvantageous, e.g. for in vivo diagnostic imaging or in situations wherethe starting polypeptide has toxic side effects when present in thecirculation for prolonged periods. Fc variants with decreased FcRnbinding affinity are also less likely to cross the placenta and, thus,are also useful in the treatment of diseases or disorders in pregnantwomen. In addition, other applications in which reduced FcRn bindingaffinity may be desired include those applications in which localizationthe brain, kidney, and/or liver is desired. In one exemplary embodiment,the chimeric protein of the invention exhibit reduced transport acrossthe epithelium of kidney glomeruli from the vasculature. In anotherembodiment, the chimeric protein of the invention exhibit reducedtransport across the blood brain barrier (BBB) from the brain, into thevascular space. In one embodiment, a protein with altered FcRn bindingcomprises at least one Fc region or FcRn binding partner (e.g, one ortwo Fc regions or FcRn binding partners) having one or more amino acidsubstitutions within the “FcRn binding loop” of an Ig constant region.The FcRn binding loop is comprised of amino acid residues 280-299(according to EU numbering) of a wild-type, full-length, Fc region. Inother embodiments, an Ig constant region or a portion thereof in achimeric protein of the invention having altered FcRn binding affinitycomprises at least one Fc region or FcRn binding partner having one ormore amino acid substitutions within the 15 {acute over (Å)} FcRn“contact zone.” As used herein, the term 15 {acute over (Å)} FcRn“contact zone” includes residues at the following positions of awild-type, full-length Fc moiety: 243-261, 275-280, 282-293, 302-319,336-348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 (EU numbering).In other embodiments, a Ig constant region or a portion thereof of theinvention having altered FcRn binding affinity comprises at least one Fcregion or FcRn binding partner having one or more amino acidsubstitutions at an amino acid position corresponding to any one of thefollowing EU positions: 256, 277-281, 283-288, 303-309, 313, 338, 342,376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplaryamino acid substitutions which altered FcRn binding activity aredisclosed in International PCT Publication No. WO05/047327 which isincorporated by reference herein.

An Fc region or FcRn binding partner used in the invention may alsocomprise an art recognized amino acid substitution which alters theglycosylation of the chimeric protein. For example, the Fc region orFcRn binding partner of the chimeric protein linked to a VWF fragment ora FVIII protein may comprise an Fc region having a mutation leading toreduced glycosylation (e.g., N- or O-linked glycosylation) or maycomprise an altered glycoform of the wild-type Fc moiety (e.g., a lowfucose or fucose-free glycan).

In one embodiment, an unprocessed chimeric protein of the invention maycomprise a genetically fused Fc region (i.e., scFc region) having two ormore of its constituent Ig constant region or a portion thereofindependently selected from the Ig constant region or a portion thereofdescribed herein. In one embodiment, the Fc regions of a dimeric Fcregion are the same. In another embodiment, at least two of the Fcregions are different. For example, the Fc regions or FcRn bindingpartners of the proteins of the invention comprise the same number ofamino acid residues or they may differ in length by one or more aminoacid residues (e.g., by about 5 amino acid residues (e.g., 1, 2, 3, 4,or 5 amino acid residues), about 10 residues, about 15 residues, about20 residues, about 30 residues, about 40 residues, or about 50residues). In yet other embodiments, the Fc regions or FcRn bindingpartners of the protein of the invention may differ in sequence at oneor more amino acid positions. For example, at least two of the Fcregions or FcRn binding partners may differ at about 5 amino acidpositions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10positions, about 15 positions, about 20 positions, about 30 positions,about 40 positions, or about 50 positions).

E) Linkers

The chimeric protein of the present invention further comprises one ormore linkers. One type of the linkers is a cleavable linker, which canbe cleaved by various proteases when administered to a subject in vivo,e.g., at a site of coagulation. In one embodiment, the cleavable linkerallows cleavage of moiety, e.g., a VWF fragment, from the chimericprotein at the site of the coagulation cascade, thus allowing activatedFVIII (FVIIIa) to have its FVIIIa activity. Another type of the linkersis a processable linker, which contains an intracellular cleavage siteand thus can be cleaved by an intracellular processing enzyme in a hostcell, allowing convenient expression of a polypeptide and formation of achimeric protein.

One or more linkers can be present between any two proteins in thechimeric protein. In one embodiment, a chimeric protein comprises (i) aVWF fragment, (ii) an XTEN sequence, and (iii) a FVIII protein, whereinthe VWF fragment is linked to the XTEN sequence by a linker, e.g., acleavable linker, and the XTEN sequence is further linked to the FVIIIprotein (i.e., V-L-X-FVIII). In another embodiment, a chimeric proteincomprises (i) a VWF fragment, (ii) an XTEN sequence, and (iii) a FVIIIprotein, wherein the VWF fragment is linked to the XTEN sequence, andthe XTEN sequence is linked to the FVIII protein by a linker, e.g., acleavable linker (i.e., V-X-L-FVIII).

In certain embodiments, a chimeric protein comprises (i) a VWF fragment,(ii) an XTEN sequence, (iii) a first Ig constant region or a portionthereof (e.g., a first Fc region), (iv) a FVIII protein, and (v) asecond Ig constant region or a portion thereof (e.g., a second Fcregion), wherein the VWF fragment is linked to the XTEN sequence by anoptional linker, e.g., a cleavable linker. The XTEN sequence can befurther linked to the first Ig constant region or a portion thereof by alinker, e.g., a cleavable linker. The FVIII protein (with or without anXTEN sequence) can also be linked to the second Ig constant region or aportion thereof by an optional linker, e.g. a cleavable linker. Incertain embodiments, the chimeric protein further comprises one or morelinkers, e.g., processable linkers, between the first Ig constant regionor a portion thereof (e.g., first Fc region) and the second Ig constantregion or a portion thereof (e.g., second Fc region), between the VWFfragment and the second Ig constant region or a portion thereof, orbetween the FVIII protein and the first Ig constant region or a portionthereof (e.g., first Fc region).

In some embodiments, the present invention includes a chimeric proteincomprising (i) a FVIII protein, (ii) an XTEN sequence, (iii) a first Igconstant region or a portion thereof, and (iv) a second Ig constantregion or a portion thereof, wherein the first Ig constant region or aportion thereof and the second Ig constant region or a portion thereofare linked by a processable linker.

The linker useful in the present invention can comprise any organicmolecule. In one embodiment, the linker comprises a polymer, e.g.,polyethylene glycol (PEG) or hydroxyethyl starch (HES). In anotherembodiment, the linker comprises an amino acids sequence. The linker cancomprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The linker cancomprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 aminoacids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids,600-700 amino acids, 700-800 amino acids, 800-900 amino acids, or900-1000 amino acids. In one embodiment, the linker comprises an XTENsequence. Additional examples of XTEN can be used according to thepresent invention and are disclosed in US Patent Publication Nos.2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1,2011/0077199 A1, or 2011/0172146 A1, or International Patent PublicationNos. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228A1, WO 2011028229 A1, or WO 2011028344 A2. In another embodiment, thelinker is a PAS sequence.

The linker useful in the present invention can comprise any organicmolecule. In one embodiment, the linker is a polymer, e.g., polyethyleneglycol (PEG) or hydroxyethyl starch (HES). In another embodiment, thelinker is an amino acid sequence. The linker can comprise at least about10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, or 2000 amino acids. The linker can comprise 1-5 amino acids, 1-10amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids,100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 aminoacids, 800-900 amino acids, or 900-1000 amino acids.

Examples of linkers are well known in the art. In one embodiment, thelinker comprises the sequence Gn. The linker can comprise the sequence(GA)_(n). The linker can comprise the sequence (GGS)_(n). In otherembodiments, the linker comprises (GGGS)_(n)(SEQ ID NO: 57). In stillother embodiments, the linker comprises the sequence(GGS)_(n)(GGGGS)_(n) (SEQ ID NO: 58). In these instances, n may be aninteger from 1-100. In other instances, n may be an integer from 1-20,i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20. Examples of linkers include, but are not limited to, GGG, SGGSGGS(SEQ ID NO: 59), GGSGGSGGSGGSGGG (SEQ ID NO: 60), GGSGGSGGGGSGGGGS (SEQID NO: 61), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 62), or GGGGSGGGGSGGGGS (SEQID NO: 63). The linker does not eliminate or diminish the VWF fragmentactivity or the clotting activity of Factor VIII. Optionally, the linkerenhances the VWF fragment activity or the clotting activity of FactorVIII protein, e.g., by further diminishing the effects of sterichindrance and making the VWF fragment or Factor VIII portion moreaccessible to its target binding site.

In one embodiment, the linker useful for the chimeric protein is 15-25amino acids long. In another embodiment, the linker useful for thechimeric protein is 15-20 amino acids long. In some embodiments, thelinker for the chimeric protein is 10-25 amino acids long. In otherembodiments, the linker for the chimeric protein is 15 amino acids long.In still other embodiments, the linker for the chimeric protein is(GGGGS)_(n) (SEQ ID NO: 64) where G represents glycine, S representsserine and n is an integer from 1-20.

F) Cleavage Sites

The linker may also incorporate a moiety capable of being cleaved eitherchemically (e.g., hydrolysis of an ester bond), enzymatically (i.e.,incorporation of a protease cleavage sequence), or photolytically (e.g.,a chromophore such as 3-amino-3-(2-nitrophenyl) proprionic acid (ANP))in order to release one molecule from another.

In one embodiment, the linker is a cleavable linker. The cleavablelinkers can comprise one or more cleavage sites at the N-terminus orC-terminus or both. In another embodiment, the cleavable linker consistsessentially of or consists of one or more cleavable sites. In otherembodiments, the cleavable linker comprises heterologous amino acidlinker sequences described herein or polymers and one or more cleavablesites.

In certain embodiments, a cleavable linker comprises one or morecleavage sites that can be cleaved in a host cell (i.e., intracellularprocessing sites). Non limiting examples of the cleavage site includeRRRR (SEQ ID NO: 9), RKRRKR (SEQ ID NO: 10), and RRRRS (SEQ ID NO: 11).

In other embodiments, a cleavable linker comprises one or more cleavagesites that are cleaved by a protease after a chimeric protein comprisingthe cleavable linker is administered to a subject. In one embodiment,the cleavage site is cleaved by a protease selected from the groupconsisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factorIXa, factor Xa, factor IIa (thrombin), Elastase-2, MMP-12, MMP-13,MMP-17, and MMP-20. In another embodiment, the cleavage site is selectedfrom the group consisting of a FXIa cleavage site (e.g., KLTR↓AET (SEQID NO: 65)), a FXIa cleavage site (e.g, DFTR↓VVG (SEQ ID NO: 66)), aFXIIa cleavage site (e.g., TMTR↓IVGG (SEQ ID NO: 67)), a Kallikreincleavage site (e.g., SPFR↓STGG (SEQ ID NO: 68)), a FVIIa cleavage site(e.g., LQVR↓IVGG (SEQ ID NO: 69)), a FIXa cleavage site (e.g., PLGR↓IVGG(SEQ ID NO: 70)), a FXa cleavage site (e.g., IEGR↓TVGG (SEQ ID NO: 71)),a FIIa (thrombin) cleavage site (e.g, LTPR↓SLLV (SEQ ID NO: 72)), aElastase-2 cleavage site (e.g, LGPV↓SGVP (SEQ ID NO: 73)), a Granzyme-Bcleavage (e.g, VAGD↓SLEE (SEQ ID NO: 74)), a MMP-12 cleavage site (e.g.,GPAG↓LGGA (SEQ ID NO: 75)), a MMP-13 cleavage site (e.g., GPAG↓LRGA (SEQID NO: 76)), a MMP-17 cleavage site (e.g., APLG↓LRLR (SEQ ID NO: 77)), aMMP-20 cleavage site (e.g., PALP↓LVAQ (SEQ ID NO: 78)), a TEV cleavagesite (e.g., ENLYFQ↓G (SEQ ID NO: 79)), a Enterokinase cleavage site(e.g., DDDK↓IVGG (SEQ ID NO: 80)), a Protease 3C (PRESCISSION™) cleavagesite (e.g., LEVLFQ↓GP (SEQ ID NO: 81)), and a Sortase A cleavage site(e.g., LPKT↓GSES) (SEQ ID NO: 82). In certain embodiments, the FXIacleavage sites include, but are not limited to, e.g., TQSFNDFTR (SEQ IDNO: 83) and SVSQTSKLTR (SEQ ID NO: 84). Non-limiting exemplary thrombincleavage sites include, e.g., DFLAEGGGVR (SEQ ID NO: 85), TTKIKPR (SEQID NO: 86), or LVPRG (SEQ ID NO: 87), and a sequence comprising,consisting essentially of, or consisting of ALRPR (SEQ ID NO: 17) (e.g.,ALRPRVVGGA (SEQ ID NO: 88)).

In a specific embodiment, the cleavage site is TLDPRSFLLRNPNDKYEPFWEDEEK(SEQ ID NO: 8).

Polynucleotides, Vectors, and Host Cells

Also provided in the invention is a polynucleotide encoding (a) a VWFfragment linked to an XTEN sequence and a FVIII protein, (b) a FVIIIprotein linked to an XTEN sequence and Fc, or (c) a FVIII protein linkedto an XTEN sequence and a VWF fragment described herein. When a chimericprotein is a single polypeptide chain (e.g., F2-L2-X-V-L1-F1-FVIII,wherein FVIII comprises a FVIII protein, F1 comprises a first Igconstant region or a portion thereof, e.g., a first Fc region, L1comprises a first linker, V comprises a VWF fragment, X comprises anXTEN sequence, L2 comprises a second linker, and F2 comprises a secondIg constant region or a portion thereof, e.g., a second Fc region), theinvention is drawn to a single polynucleotide chain encoding the singlepolypeptide chain. When the chimeric protein comprises a first and asecond polypeptide chains (F2-L2-X-V:FVIII-F1), the first polypeptidechain comprising a VWF fragment linked to a XTEN sequence, which isfurther linked to a first Ig constant region or a portion thereof (e.g.,a first Fc region) by a cleavable linker (e.g., F2-L2-X-V) and thesecond polypeptide chain comprising a FVIII protein and a second Igconstant region or a portion thereof (e.g., a second Fc region) (e.g,FVIII-F1), wherein the first polypeptide chain and the secondpolypeptide chain are associated with each other, a polynucleotide cancomprise the first nucleotide sequence and the second nucleotidesequence. In one embodiment, the first polypeptide chain and the secondpolypeptide chain can be encoded by a single polynucleotide chain. Inanother embodiment, the first polypeptide chain and the secondpolypeptide chain are encoded by two different polynucleotides, i.e., afirst nucleotide sequence and a second nucleotide sequence. In anotherembodiment, the first nucleotide sequence and the second nucleotidesequence are on two different polynucleotides (e.g., different vectors).In certain embodiments, the present invention is directed to a set ofpolynucleotides comprising a first nucleotide chain and a secondnucleotide chain, wherein the first nucleotide chain encodes the VWFfragment of the chimeric protein and the second nucleotide chain encodesthe FVIII protein. In some embodiments, a chimeric protein comprisingtwo polypeptide chains or three polypeptide chains can be encoded by asingle polynucleotide chain, and then processed into two or three (ormore) polypeptide chains. In yet other embodiments, a chimeric proteincomprising these polypeptide chains can be encoded by two or threepolynucleotide chains.

In other embodiments, the set of the polynucleotides further comprisesan additional nucleotide chain (e.g., a second nucleotide chain when thechimeric polypeptide is encoded by a single polynucleotide chain or athird nucleotide chain when the chimeric protein is encoded by twopolynucleotide chains) which encodes a protein convertase. The proteinconvertase can be selected from the group consisting of proproteinconvertase subtilisin/kexin type 5 (PCSK5 or PC5), proprotein convertasesubtilisin/kexin type 7 (PCSK7 or PC5), a yeast Kex 2, proproteinconvertase subtilisin/kexin type 3 (PACE or PCSK3), and two or morecombinations thereof. In some embodiments, the protein convertase isPACE, PC5, or PC7. In a specific embodiment, the protein convertase isPC5 or PC7. See International Application no. PCT/US2011/043568.

As used herein, an expression vector refers to any nucleic acidconstruct which contains the necessary elements for the transcriptionand translation of an inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation,when introduced into an appropriate host cell. Expression vectors caninclude plasmids, phagemids, viruses, and derivatives thereof.

Expression vectors of the invention will include polynucleotidesencoding the chimeric protein described herein. In one embodiment, oneor more of the coding sequences for the VWF fragment and XTEN, the FVIIIprotein and XTEN, or both are operably linked to an expression controlsequence. As used herein, two nucleic acid sequences are operably linkedwhen they are covalently linked in such a way as to permit eachcomponent nucleic acid sequence to retain its functionality. A codingsequence and a gene expression control sequence are said to be operablylinked when they are covalently linked in such a way as to place theexpression or transcription and/or translation of the coding sequenceunder the influence or control of the gene expression control sequence.Two DNA sequences are said to be operably linked if induction of apromoter in the 5′ gene expression sequence results in the transcriptionof the coding sequence and if the nature of the linkage between the twoDNA sequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the promoter region todirect the transcription of the coding sequence, or (3) interfere withthe ability of the corresponding RNA transcript to be translated into aprotein. Thus, a gene expression sequence would be operably linked to acoding nucleic acid sequence if the gene expression sequence werecapable of effecting transcription of that coding nucleic acid sequencesuch that the resulting transcript is translated into the desiredprotein or polypeptide.

A gene expression control sequence as used herein is any regulatorynucleotide sequence, such as a promoter sequence or promoter-enhancercombination, which facilitates the efficient transcription andtranslation of the coding nucleic acid to which it is operably linked.The gene expression control sequence may, for example, be a mammalian orviral promoter, such as a constitutive or inducible promoter.Constitutive mammalian promoters include, but are not limited to, thepromoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actinpromoter, and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40),papilloma virus, adenovirus, human immunodeficiency virus (HIV), Roussarcoma virus, cytomegalovirus, the long terminal repeats (LTR) ofMoloney leukemia virus, and other retroviruses, and the thymidine kinasepromoter of herpes simplex virus. Other constitutive promoters are knownto those of ordinary skill in the art. The promoters useful as geneexpression sequences of the invention also include inducible promoters.Inducible promoters are expressed in the presence of an inducing agent.For example, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

In general, the gene expression control sequence shall include, asnecessary, 5′ non-transcribing and 5′ non-translating sequences involvedwith the initiation of transcription and translation, respectively, suchas a TATA box, capping sequence, CAAT sequence, and the like.Especially, such 5′ non-transcribing sequences will include a promoterregion which includes a promoter sequence for transcriptional control ofthe operably joined coding nucleic acid. The gene expression sequencesoptionally include enhancer sequences or upstream activator sequences asdesired.

Viral vectors include, but are not limited to, nucleic acid sequencesfrom the following viruses: retrovirus, such as Moloney murine leukemiavirus, Harvey murine sarcoma virus, murine mammary tumor virus, and Roussarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses;polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus;vaccinia virus; polio virus; and RNA virus such as a retrovirus. One canreadily employ other vectors well-known in the art. Certain viralvectors are based on non-cytopathic eukaryotic viruses in whichnon-essential genes have been replaced with the gene of interest.Non-cytopathic viruses include retroviruses, the life cycle of whichinvolves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. Retroviruseshave been approved for human gene therapy trials. Most useful are thoseretroviruses that are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell line withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H.Freeman Co., New York (1990) and Murry, E. J., Methods in MolecularBiology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

In one embodiment, the virus is an adeno-associated virus, adouble-stranded DNA virus. The adeno-associated virus can be engineeredto be replication-deficient and is capable of infecting a wide range ofcell types and species. It further has advantages such as heat and lipidsolvent stability; high transduction frequencies in cells of diverselineages, including hematopoietic cells; and lack of superinfectioninhibition thus allowing multiple series of transductions. Reportedly,the adeno-associated virus can integrate into human cellular DNA in asite-specific manner, thereby minimizing the possibility of insertionalmutagenesis and variability of inserted gene expression characteristicof retroviral infection. In addition, wild-type adeno-associated virusinfections have been followed in tissue culture for greater than 100passages in the absence of selective pressure, implying that theadeno-associated virus genomic integration is a relatively stable event.The adeno-associated virus can also function in an extrachromosomalfashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well-known to those of skill inthe art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been found to be particularlyadvantageous for delivering genes to cells in vivo because of theirinability to replicate within and integrate into a host genome. Theseplasmids, however, having a promoter compatible with the host cell, canexpress a peptide from a gene operably encoded within the plasmid. Somecommonly used plasmids available from commercial suppliers includepBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMVplasmids, pSV40, and pBlueScript. Additional examples of specificplasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro,catalog number V87020; pcDNA4/myc-His, catalog number V86320; andpBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad,Calif.). Other plasmids are well-known to those of ordinary skill in theart. Additionally, plasmids may be custom designed using standardmolecular biology techniques to remove and/or add specific fragments ofDNA.

In one insect expression system that may be used to produce the proteinsof the invention, Autographa californica nuclear polyhidrosis virus(AcNPV) is used as a vector to express the foreign genes. The virusgrows in Spodoptera frugiperda cells. A coding sequence may be clonedinto non-essential regions (for example, the polyhedron gene) of thevirus and placed under control of an ACNPV promoter (for example, thepolyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (see, e.g., Smith et al. (1983) J Virol 46:584; U.S.Pat. No. 4,215,051). Further examples of this expression system may befound in Ausubel et al., eds. (1989) Current Protocols in MolecularBiology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

Another system which can be used to express the proteins of theinvention is the glutamine synthetase gene expression system, alsoreferred to as the “GS expression system” (Lonza Biologics PLC,Berkshire UK). This expression system is described in detail in U.S.Pat. No. 5,981,216.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. See, e.g., Logan & Shenk (1984) Proc NatlAcad Sci USA 81:3655). Alternatively, the vaccinia 7.5 K promoter may beused. See, e.g., Mackett et al. (1982) Proc Natl Acad Sci USA 79:7415;Mackett et al. (1984) J Virol 49:857; Panicali et al. (1982) Proc NatlAcad Sci USA 79:4927.

To increase efficiency of production, the polynucleotides can bedesigned to encode multiple units of the protein of the inventionseparated by enzymatic cleavage sites. The resulting polypeptide can becleaved (e.g., by treatment with the appropriate enzyme) in order torecover the polypeptide units. This can increase the yield ofpolypeptides driven by a single promoter. When used in appropriate viralexpression systems, the translation of each polypeptide encoded by themRNA is directed internally in the transcript; e.g., by an internalribosome entry site, IRES. Thus, the polycistronic construct directs thetranscription of a single, large polycistronic mRNA which, in turn,directs the translation of multiple, individual polypeptides. Thisapproach eliminates the production and enzymatic processing ofpolyproteins and may significantly increase the yield of polypeptidesdriven by a single promoter.

Vectors used in transformation will usually contain a selectable markerused to identify transformants. In bacterial systems, this can includean antibiotic resistance gene such as ampicillin or kanamycin.Selectable markers for use in cultured mammalian cells include genesthat confer resistance to drugs, such as neomycin, hygromycin, andmethotrexate. The selectable marker may be an amplifiable selectablemarker. One amplifiable selectable marker is the dihydrofolate reductase(DHFR) gene. Simonsen C C et al. (1983) Proc Natl Acad Sci USA80:2495-9. Selectable markers are reviewed by Thilly (1986) MammalianCell Technology, Butterworth Publishers, Stoneham, Mass., and the choiceof selectable markers is well within the level of ordinary skill in theart.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, U.S. Pat. No.4,713,339).

The expression vectors can encode for tags that permit easy purificationof the recombinantly produced protein. Examples include, but are notlimited to, vector pUR278 (Ruther et al. (1983) EMBO J 2:1791), in whichcoding sequences for the protein to be expressed may be ligated into thevector in frame with the lac z coding region so that a tagged fusionprotein is produced; pGEX vectors may be used to express proteins of theinvention with a glutathione S-transferase (GST) tag. These proteins areusually soluble and can easily be purified from cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The vectors include cleavage sites (thrombin or Factor Xaprotease or PRESCISSION PROTEASE™ (Pharmacia, Peapack, N.J.)) for easyremoval of the tag after purification.

The expression vector or vectors are then transfected or co-transfectedinto a suitable target cell, which will express the polypeptides.Transfection techniques known in the art include, but are not limitedto, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725),electroporation (Neumann et al. (1982) EMBO J 1:841), and liposome-basedreagents. A variety of host-expression vector systems may be utilized toexpress the proteins described herein including both prokaryotic andeukaryotic cells. These include, but are not limited to, microorganismssuch as bacteria (e.g., E. coli) transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems, including mammalian cells (e.g., HEK 293, CHO, Cos, HeLa,HKB11, and BHK cells).

In one embodiment, the host cell is a eukaryotic cell. As used herein, aeukaryotic cell refers to any animal or plant cell having a definitivenucleus. Eukaryotic cells of animals include cells of vertebrates, e.g.,mammals, and cells of invertebrates, e.g., insects. Eukaryotic cells ofplants specifically can include, without limitation, yeast cells. Aeukaryotic cell is distinct from a prokaryotic cell, e.g., bacteria.

In certain embodiments, the eukaryotic cell is a mammalian cell. Amammalian cell is any cell derived from a mammal. Mammalian cellsspecifically include, but are not limited to, mammalian cell lines. Inone embodiment, the mammalian cell is a human cell. In anotherembodiment, the mammalian cell is a HEK 293 cell, which is a humanembryonic kidney cell line. HEK 293 cells are available as CRL-1533 fromAmerican Type Culture Collection, Manassas, Va., and as 293-H cells,Catalog No. 11631-017 or 293-F cells, Catalog No. 11625-019 fromInvitrogen (Carlsbad, Calif.). In some embodiments, the mammalian cellis a PER.C6® cell, which is a human cell line derived from retina.PER.C6® cells are available from Crucell (Leiden, The Netherlands). Inother embodiments, the mammalian cell is a Chinese hamster ovary (CHO)cell. CHO cells are available from American Type Culture Collection,Manassas, Va. (e.g., CHO-K1; CCL-61). In still other embodiments, themammalian cell is a baby hamster kidney (BHK) cell. BHK cells areavailable from American Type Culture Collection, Manassas, Va. (e.g.,CRL-1632). In some embodiments, the mammalian cell is a HKB11 cell,which is a hybrid cell line of a HEK293 cell and a human B cell line.Mei et al., Mol. Biotechnol. 34(2): 165-78 (2006).

In one embodiment, a plasmid including a FVIII(X)-Fc fusion codingsequence, a VWF fragment-L-Fc fusion coding sequence, or both and aselectable marker, e.g., zeocin resistance, are transfected into HEK 293cells, for production of a chimeric protein.

In another embodiment, a plasmid including a FVIII-Fc fusion codingsequence, a VWF fragment-XTEN-L-Fc fusion coding sequence, or both and aselectable marker, e.g., zeocin resistance, are transfected into HEK 293cells, for production of a chimeric protein.

In other embodiments, a plasmid including a FVIII(X)-Fc fusion codingsequence, a Fc coding sequence, or both and a selectable marker, e.g.,zeocin resistance, are transfected into HEK 293 cells, for production ofa chimeric protein.

In some embodiments, a first plasmid including a FVIII(X)-Fc fusioncoding sequence and a first selectable marker, e.g., a zeocin resistancegene, and a second plasmid including an Fc coding sequence or a VWFfragment-L-Fc coding sequence and a second selectable marker, e.g., aneomycin resistance gene, and a third plasmid including a proteinconvertase coding sequence and a third selectable marker, e.g., ahygromycin resistance gene, are cotransfected into HEK 293 cells, forproduction of the chimeric protein. The first and second plasmids can beintroduced in equal amounts (i.e., 1:1 molar ratio), or they can beintroduced in unequal amounts.

In still other embodiments, a first plasmid including a FVIII-Fc fusioncoding sequence and a first selectable marker, e.g., a zeocin resistancegene, and a second plasmid including a VWF fragment-XTEN-L-Fc codingsequence and a second selectable marker, e.g., a neomycin resistancegene, and a third plasmid including a protein convertase coding sequenceand a third selectable marker, e.g., a hygromycin resistance gene, arecotransfected into HEK 293 cells, for production of the chimericprotein. The first and second plasmids can be introduced in equalamounts (i.e., 1:1 molar ratio), or they can be introduced in unequalamounts.

In yet other embodiments, a first plasmid including a FVIII(X)-Fc fusioncoding sequence and a first selectable marker, e.g., a zeocin resistancegene, and a second plasmid including a VWF fragment-XTEN-L-Fc codingsequence and a second selectable marker, e.g., a neomycin resistancegene, and a third plasmid including a protein convertase coding sequenceand a third selectable marker, e.g., a hygromycin resistance gene, arecotransfected into HEK 293 cells, for production of the chimericprotein. The first and second plasmids can be introduced in equalamounts (i.e., 1:1 molar ratio), or they can be introduced in unequalamounts.

In certain embodiments, a first plasmid, including a chimeric proteinencoding FVIII (with or without XTEN)-F1-L1-V-XTEN-L2-F2 coding sequenceand a first selectable marker, e.g., a zeocin resistance gene, and asecond plasmid including a protein convertase coding sequence and asecond selectable marker, e.g., a hygromycin resistance gene, arecotransfected into HEK 293 cells, for production of the chimericprotein. The promoters for the FVIII(X)-Fc coding sequence and theVWF-XTEN-Fc coding sequence can be different or they can be the same.

In still other embodiments, transfected cells are stably transfected.These cells can be selected and maintained as a stable cell line, usingconventional techniques known to those of skill in the art.

Host cells containing DNA constructs of the protein are grown in anappropriate growth medium. As used herein, the term “appropriate growthmedium” means a medium containing nutrients required for the growth ofcells. Nutrients required for cell growth may include a carbon source, anitrogen source, essential amino acids, vitamins, minerals, and growthfactors. Optionally, the media can contain one or more selectionfactors. Optionally the media can contain bovine calf serum or fetalcalf serum (FCS). In one embodiment, the media contains substantially noIgG. The growth medium will generally select for cells containing theDNA construct by, for example, drug selection or deficiency in anessential nutrient which is complemented by the selectable marker on theDNA construct or co-transfected with the DNA construct. Culturedmammalian cells are generally grown in commercially availableserum-containing or serum-free media (e.g., MEM, DMEM, DMEM/F12). In oneembodiment, the medium is CD293 (Invitrogen, Carlsbad, Calif.). Inanother embodiment, the medium is CD17 (Invitrogen, Carlsbad, Calif.).Selection of a medium appropriate for the particular cell line used iswithin the level of those ordinary skilled in the art.

In order to co-express the two polypeptide chains of the chimericprotein, the host cells are cultured under conditions that allowexpression of both chains. As used herein, culturing refers tomaintaining living cells in vitro for at least a definite time.Maintaining can, but need not include, an increase in population ofliving cells. For example, cells maintained in culture can be static inpopulation, but still viable and capable of producing a desired product,e.g., a recombinant protein or recombinant fusion protein. Suitableconditions for culturing eukaryotic cells are well known in the art andinclude appropriate selection of culture media, media supplements,temperature, pH, oxygen saturation, and the like. For commercialpurposes, culturing can include the use of any of various types ofscale-up systems including shaker flasks, roller bottles, hollow fiberbioreactors, stirred-tank bioreactors, airlift bioreactors, Wavebioreactors, and others.

The cell culture conditions are also selected to allow association ofthe VWF fragment with the FVIII protein. Conditions that allowexpression of the VWF fragment and/or the FVIII protein may include thepresence of a source of vitamin K. For example, in one embodiment,stably transfected HEK 293 cells are cultured in CD293 media(Invitrogen, Carlsbad, Calif.) or OptiCHO media (Invitrogen, Carlsbad,Calif.) supplemented with 4 mM glutamine.

In one aspect, the present invention is directed to a method ofexpressing, making, or producing the chimeric protein of the inventioncomprising a) transfecting a host cell comprising a polynucleotideencoding the chimeric protein and b) culturing the host cell in aculture medium under a condition suitable for expressing the chimericprotein, wherein the chimeric protein is expressed.

In further embodiments, the protein product containing the VWF fragmentlinked to an XTEN sequence or the FVIII protein linked to an XTENsequence is secreted into the media. Media is separated from the cells,concentrated, filtered, and then passed over two or three affinitycolumns, e.g., a protein A column and one or two anion exchange columns.

In certain aspects, the present invention relates to the chimericprotein produced by the methods described herein.

In vitro production allows scale-up to give large amounts of the desiredaltered polypeptides of the invention. Techniques for mammalian cellcultivation under tissue culture conditions are known in the art andinclude homogeneous suspension culture, e.g. in an airlift reactor or ina continuous stirrer reactor, or immobilized or entrapped cell culture,e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, hydrophobic interactionchromatography (HIC, chromatography over DEAE-cellulose or affinitychromatography.

Pharmaceutical Composition

Compositions containing the chimeric protein of the present inventionmay contain a suitable pharmaceutically acceptable carrier. For example,they may contain excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations designed fordelivery to the site of action.

The pharmaceutical composition can be formulated for parenteraladministration (i.e. intravenous, subcutaneous, or intramuscular) bybolus injection. Formulations for injection can be presented in unitdosage form, e.g., in ampoules or in multidose containers with an addedpreservative. The compositions can take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., pyrogen free water.

Suitable formulations for parenteral administration also include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substances,which increase the viscosity of the suspension, including, for example,sodium carboxymethyl cellulose, sorbitol and dextran. Optionally, thesuspension may also contain stabilizers. Liposomes also can be used toencapsulate the molecules of the invention for delivery into cells orinterstitial spaces. Exemplary pharmaceutically acceptable carriers arephysiologically compatible solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, water, saline, phosphate buffered saline, dextrose, glycerol,ethanol and the like. In some embodiments, the composition comprisesisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride. In other embodiments, the compositionscomprise pharmaceutically acceptable substances such as wetting agentsor minor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the active ingredients.

Compositions of the invention may be in a variety of forms, including,for example, liquid (e.g., injectable and infusible solutions),dispersions, suspensions, semi-solid and solid dosage forms. Thepreferred form depends on the mode of administration and therapeuticapplication.

The composition can be formulated as a solution, micro emulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared byincorporating the active ingredient in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active ingredient into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution. Theproper fluidity of a solution can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The active ingredient can be formulated with a controlled-releaseformulation or device. Examples of such formulations and devices includeimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, for example, ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for the preparation ofsuch formulations and devices are known in the art. See e.g., Sustainedand Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,Marcel Dekker, Inc., New York, 1978.

Injectable depot formulations can be made by forming microencapsulatedmatrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the polymer employed, the rate of drug release can becontrolled. Other exemplary biodegradable polymers are polyorthoestersand polyanhydrides. Depot injectable formulations also can be preparedby entrapping the drug in liposomes or microemulsions.

Supplementary active compounds can be incorporated into thecompositions. In one embodiment, the chimeric protein of the inventionis formulated with another clotting factor, or a variant, fragment,analogue, or derivative thereof. For example, the clotting factorincludes, but is not limited to, factor V, factor VII, factor VIII,factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin,fibrinogen, von Willebrand factor or recombinant soluble tissue factor(rsTF) or activated forms of any of the preceding. The clotting factorof hemostatic agent can also include anti-fibrinolytic drugs, e.g.,epsilon-amino-caproic acid, tranexamic acid.

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time, or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.See, e.g., Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton,Pa. 1980).

In addition to the active compound, the liquid dosage form may containinert ingredients such as water, ethyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan.

Non-limiting examples of suitable pharmaceutical carriers are alsodescribed in Remington's Pharmaceutical Sciences by E. W. Martin. Someexamples of excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, and the like. The composition canalso contain pH buffering reagents, and wetting or emulsifying agents.

For oral administration, the pharmaceutical composition can take theform of tablets or capsules prepared by conventional means. Thecomposition can also be prepared as a liquid for example a syrup or asuspension. The liquid can include suspending agents (e.g., sorbitolsyrup, cellulose derivatives or hydrogenated edible fats), emulsifyingagents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil,oily esters, ethyl alcohol, or fractionated vegetable oils), andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). The preparations can also include flavoring, coloring andsweetening agents. Alternatively, the composition can be presented as adry product for constitution with water or another suitable vehicle.

For buccal administration, the composition may take the form of tabletsor lozenges according to conventional protocols.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of a nebulizedaerosol with or without excipients or in the form of an aerosol sprayfrom a pressurized pack or nebulizer, with optionally a propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator can be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The pharmaceutical composition can also be formulated for rectaladministration as a suppository or retention enema, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

In one embodiment, a pharmaceutical composition comprises a chimericprotein, the polynucleotide encoding the chimeric protein, the vectorcomprising the polynucleotide, or the host cell comprising the vector,and a pharmaceutically acceptable carrier. The FVIII protein in achimeric protein has extended half-life compared to wild type FVIIIprotein or the corresponding FVIII protein without the VWF fragment. Inone embodiment, wherein the half-life of the FVIII protein is extendedat least about 1.5 times, at least about 2 times, at least about 2.5times, at least about 3 times, at least about 4 times, at least about 5times, at least about 6 times, at least about 7 times, at least about 8times, at least about 9 times, at least about 10 times, at least about11 times, or at least about 12 times longer than wild type FVIII. Inanother embodiment, the half-life of Factor VIII is at least about 17hours, at least about 18 hours, at least about 19 hours, at least about20 hours, at least about 21 hours, at least about 22 hours, at leastabout 23 hours, at least about 24 hours, at least about 25 hours, atleast about 26 hours, at least about 27 hours, at least about 28 hours,at least about 29 hours, at least about 30 hours, at least about 31hours, at least about 32 hours, at least about 33 hours, at least about34 hours, at least about 35 hours, at least about 36 hours, at leastabout 48 hours, at least about 60 hours, at least about 72 hours, atleast about 84 hours, at least about 96 hours, or at least about 108hours.

In some embodiments, the composition is administered by a route selectedfrom the group consisting of topical administration, intraocularadministration, parenteral administration, intrathecal administration,subdural administration and oral administration. The parenteraladministration can be intravenous or subcutaneous administration.

In other embodiments, the composition is used to treat a bleedingdisease or condition in a subject in need thereof. The bleeding diseaseor condition is selected from the group consisting of a bleedingcoagulation disorder, hemarthrosis, muscle bleed, oral bleed,hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, traumacapitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, bleeding in the illiopsoas sheathand any combinations thereof. In still other embodiments, the subject isscheduled to undergo a surgery. In yet other embodiments, the treatmentis prophylactic or on-demand.

Gene Therapy

A chimeric protein thereof of the invention can be produced in vivo in amammal, e.g., a human patient, using a gene therapy approach totreatment of a bleeding disease or disorder selected from the groupconsisting of a bleeding coagulation disorder, hemarthrosis, musclebleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage,trauma, trauma capitis, gastrointestinal bleeding, intracranialhemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bonefracture, central nervous system bleeding, bleeding in theretropharyngeal space, bleeding in the retroperitoneal space, andbleeding in the illiopsoas sheath would be therapeutically beneficial.In one embodiment, the bleeding disease or disorder is hemophilia. Inanother embodiment, the bleeding disease or disorder is hemophilia A.This involves administration of a suitable chimeric protein-encodingnucleic acid operably linked to suitable expression control sequences.In certain embodiment, these sequences are incorporated into a viralvector. Suitable viral vectors for such gene therapy include adenoviralvectors, lentiviral vectors, baculoviral vectors, Epstein Barr viralvectors, papovaviral vectors, vaccinia viral vectors, herpes simplexviral vectors, and adeno associated virus (AAV) vectors. The viralvector can be a replication-defective viral vector. In otherembodiments, an adenoviral vector has a deletion in its E1 gene or E3gene. When an adenoviral vector is used, the mammal may not be exposedto a nucleic acid encoding a selectable marker gene. In otherembodiments, the sequences are incorporated into a non-viral vectorknown to those skilled in the art.

Methods of Using Chimeric Protein

The present invention is directed to a method of using a chimericprotein described herein to prevent or inhibit endogenous VWF binding toa FVIII protein. The present invention is also directed to a method ofusing a chimeric protein having a FVIII protein linked to XTEN and an Igconstant region or a portion thereof.

One aspect of the present invention is directed to preventing orinhibiting FVIII interaction with endogenous VWF by blocking orshielding the VWF binding site on the FVIII from endogenous VWF and atthe same time extending half-life of the FVIII protein using an XTENsequence in combination with an Ig constant region or a portion thereof,which can also be a half-life extender. In one embodiment, the inventionis directed to a method of constructing a FVIII protein having half-lifelonger than wild-type FVIII. In one embodiment, an XTEN sequenceinhibits or prevents interaction of a FVIII protein in a chimericprotein with endogenous VWF. In another embodiment, an Ig constantregion or a portion thereof inhibits or prevents interaction of theFVIII protein with endogenous VWF. The chimeric protein useful in themethod includes any one or more chimeric protein described herein.

Another aspect of the invention includes a method of administering to asubject in need thereof a chimeric protein comprising a FVIII proteinhaving half-life longer than wild-type FVIII, wherein the methodcomprises administering the chimeric protein described herein to thesubject.

In one embodiment, the invention is directed to a method of using anXTEN sequence and an Ig constant region or a portion thereof to extend ahalf-life of a FVIII protein and a VWF fragment to prevent or inhibitendogenous VWF interaction with a FVIII protein. A FVIII protein linkedto an XTEN sequence (e.g., FVIII(X)) and then bound to or associatedwith a VWF fragment is shielded or protected from the clearance pathwayof VWF and thus has reduced clearance compared to the FVIII protein notbound to the VWF fragment. The shielded FVIII protein thus has maximumextension of a half-life compared to a FVIII protein not bound to orassociated with the XTEN sequence and the VWF fragment. In certainembodiments, the FVIII protein associated with or protected by a VWFfragment and linked to an XTEN sequence is not cleared by a VWFclearance receptor. In other embodiments, the FVIII protein associatedwith or protected by a VWF fragment and linked to an XTEN sequence iscleared from the system slower than the FVIII protein that is notassociated with or protected by the VWF fragment and linked to the XTENsequence.

In one aspect, the chimeric protein comprising the FVIII protein linkedto an XTEN sequence or the FVIII protein bound to or associated with aVWF fragment linked to XTEN has reduced clearance from circulation asthe VWF fragment does not contain a VWF clearance receptor binding site.The VWF fragment prevents or inhibits clearance of FVIII bound to orassociated with the VWF fragment from the system through the VWFclearance pathway. The VWF fragments useful for the present inventioncan also provide at least one or more VWF-like FVIII protectionproperties that are provided by endogenous VWF. In certain embodiments,the VWF fragment or the XTEN sequence can also mask one or more FVIIIclearance receptor binding site, thereby preventing clearance of FVIIIby its own clearance pathway.

In some embodiments, the prevention or inhibition of a FVIII proteinbinding to endogenous VWF by the VWF fragment or the XTEN sequence canbe in vitro or in vivo.

Also provided is a method of increasing the half-life of a FVIII proteincomprising administering the chimeric protein described herein to asubject in need thereof. The half-life of non-activated FVIII bound toor associated with full-length VWF is about 12 to 14 hours in plasma. InVWD type 3, wherein there is almost no VWF in circulation, the half-lifeof FVIII is only about six hours, leading to symptoms of mild tomoderate hemophilia A in such patients due to decreased concentrationsof FVIII. The half-life of the FVIII protein linked to or associatedwith the VWF fragment or the XTEN sequence of the present invention canincrease at least about 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.6 times,2.7. times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times,or 4.0 times higher than the half-life of the non-activated FVIII boundto or associated with full-length VWF.

In one embodiment, the half-life of the FVIII protein linked to orassociated with the VWF fragment or linked to an Ig constant region or aportion thereof in the chimeric protein comprising an XTEN sequenceincreases at least about 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 7 times, 8 times, 9times, or 10 times higher than the half-life of the non-activated FVIIIbound to or associated with full-length VWF. In another embodiment, thehalf-life of the FVIII protein linked to or associated with the VWFfragment or an Ig constant region or a portion thereof in the chimericprotein comprising an XTEN sequence increases about 2 to about 5 times,about 3 to about 10 times, about 5 to about 15 times, about 10 to about20 times, about 15 to about 25 times, about 20 to about 30 times, about25 to about 35 times, about 30 to about 40 times, about 35 to about 45times higher than the half-life of the non-activated FVIII bound to orassociated with full-length VWF or wild type FVIII. In a specificembodiment, the half-life of the FVIII protein linked to or associatedwith the VWF fragment or linked to an Ig constant region in the chimericprotein comprising an XTEN sequence increases at least about 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 times higher than the half-life of thewild type FVIII in a FVIII and VWF double knockout mouse.

In some embodiments, the half-life of the chimeric protein comprisingthe VWF fragment fused to a first Ig constant region or a portionthereof, e.g., a first Fc region and an XTEN sequence, and a FVIIIprotein linked to an XTEN sequence and a second Ig constant region or aportion thereof, e.g., a second Fc region, is longer than the half-lifeof a FVIII associated with endogenous VWF. In other embodiments, thehalf-life of the chimeric protein is at least about 1.5 times, 2 times,2.5 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0times, 4.5 times, or 5.0 times the half-life of wild type FVIII or aFVIII protein associated with endogenous VWF.

In some embodiments, as a result of the invention the half-life of theFVIII protein is extended compared to a FVIII protein without the VWFfragment or wild-type FVIII. The half-life of the chimeric protein ofthe invention is at least about 1.5 times, at least about 2 times, atleast about 2.5 times, at least about 3 times, at least about 4 times,at least about 5 times, at least about 6 times, at least about 7 times,at least about 8 times, at least about 9 times, at least about 10 times,at least about 11 times, or at least about 12 times longer than thehalf-life of a FVIII protein without the VWF fragment or wild-typeFVIII. In one embodiment, the half-life of FVIII is about 1.5-fold toabout 20-fold, about 1.5 fold to about 15 fold, or about 1.5 fold toabout 10 fold longer than the half-life of wild-type FVIII. In anotherembodiment, the half-life of the FVIII is extended about 2-fold to about10-fold, about 2-fold to about 9-fold, about 2-fold to about 8-fold,about 2-fold to about 7-fold, about 2-fold to about 6-fold, about 2-foldto about 5-fold, about 2-fold to about 4-fold, about 2-fold to about3-fold, about 2.5-fold to about 10-fold, about 2.5-fold to about 9-fold,about 2.5-fold to about 8-fold, about 2.5-fold to about 7-fold, about2.5-fold to about 6-fold, about 2.5-fold to about 5-fold, about 2.5-foldto about 4-fold, about 2.5-fold to about 3-fold, about 3-fold to about10-fold, about 3-fold to about 9-fold, about 3-fold to about 8-fold,about 3-fold to about 7-fold, about 3-fold to about 6-fold, about 3-foldto about 5-fold, about 3-fold to about 4-fold, about 4-fold to about 6fold, about 5-fold to about 7-fold, or about 6-fold to about 8 fold ascompared to wild-type FVIII or a FVIII protein without the VWF fragment.In other embodiments, the half-life of the chimeric protein of theinvention is at least about 17 hours, at least about 18 hours, at leastabout 19 hours, at least about 20 hours, at least about 21 hours, atleast about 22 hours, at least about 23 hours, at least about 24 hours,at least about 25 hours, at least about 26 hours, at least about 27hours, at least about 28 hours, at least about 29 hours, at least about30 hours, at least about 31 hours, at least about 32 hours, at leastabout 33 hours, at least about 34 hours, at least about 35 hours, atleast about 36 hours, at least about 48 hours, at least about 60 hours,at least about 72 hours, at least about 84 hours, at least about 96hours, or at least about 108 hours. In still other embodiments, thehalf-life of the chimeric protein of the invention is about 15 hours toabout two weeks, about 16 hours to about one week, about 17 hours toabout one week, about 18 hours to about one week, about 19 hours toabout one week, about 20 hours to about one week, about 21 hours toabout one week, about 22 hours to about one week, about 23 hours toabout one week, about 24 hours to about one week, about 36 hours toabout one week, about 48 hours to about one week, about 60 hours toabout one week, about 24 hours to about six days, about 24 hours toabout five days, about 24 hours to about four days, about 24 hours toabout three days, or about 24 hours to about two days.

In some embodiments, the average half-life of the chimeric protein ofthe invention per subject is about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, about 24 hours (1 day), about 25 hours,about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours,about 35 hours, about 36 hours, about 40 hours, about 44 hours, about 48hours (2 days), about 54 hours, about 60 hours, about 72 hours (3 days),about 84 hours, about 96 hours (4 days), about 108 hours, about 120hours (5 days), about six days, about seven days (one week), about eightdays, about nine days, about 10 days, about 11 days, about 12 days,about 13 days, or about 14 days.

In addition, the invention provides a method of treating or preventing ableeding disease or disorder comprising administering an effectiveamount of a chimeric protein. In one embodiment, the bleeding disease ordisorder is selected from the group consisting of a bleeding coagulationdisorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhageinto muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinalbleeding, intracranial hemorrhage, intra-abdominal hemorrhage,intrathoracic hemorrhage, bone fracture, central nervous systembleeding, bleeding in the retropharyngeal space, bleeding in theretroperitoneal space, and bleeding in the illiopsoas sheath. In aspecific embodiment, the bleeding disease or disorder is hemophilia A.

The chimeric protein comprising an XTEN sequence and an Ig constantregion or a portion thereof in combination with a VWF fragment describedherein, that prevents or inhibits interaction of the FVIII protein withendogenous VWF prepared by the invention, has many uses as will berecognized by one skilled in the art, including, but not limited tomethods of treating a subject having a hemostatic disorder and methodsof treating a subject in need of a general hemostatic agent. In oneembodiment, the invention relates to a method of treating a subjecthaving a hemostatic disorder comprising administering a therapeuticallyeffective amount of the chimeric protein.

The FVIII protein portion in the chimeric protein treats or prevents ahemostatic disorder by serving as a cofactor to Factor IX on anegatively charged phospholipid surface, thereby forming a Xase complex.The binding of activated coagulation factors to a phospholipid surfacelocalizes this process to sites of vascular damage. On a phospholipidsurface, Factor VIIIa increases the maximum velocity of Factor Xactivation by Factor IXa, by approximately 200,000-fold, leading to thelarge second burst of thrombin generation.

The chimeric protein of the invention can be used to treat anyhemostatic disorder. The hemostatic disorders that may be treated byadministration of the chimeric protein of the invention include, but arenot limited to, hemophilia A, as well as deficiencies or structuralabnormalities relating to Factor VIII. In one embodiment, the hemostaticdisorder is hemophilia A.

The chimeric protein of the invention can be used prophylactically totreat a subject with a hemostatic disorder. The chimeric protein of theinvention can be used to treat an acute bleeding episode in a subjectwith a hemostatic disorder. In another embodiment, the hemostaticdisorder can be the result of a defective clotting factor, e.g., vonWillebrand's factor. In one embodiment, the hemostatic disorder is aninherited disorder. In another embodiment, the hemostatic disorder is anacquired disorder. The acquired disorder can result from an underlyingsecondary disease or condition. The unrelated condition can be, as anexample, but not as a limitation, cancer, an auto-immune disease, orpregnancy. The acquired disorder can result from old age or frommedication to treat an underlying secondary disorder (e.g. cancerchemotherapy).

The invention also relates to methods of treating a subject that doesnot have a congenital hemostatic disorder, but has a secondary diseaseor condition resulting in acquisition of a hemostatic disorder, e.g.,due to development of an anti-FVIII antibody or a surgery. The inventionthus relates to a method of treating a subject in need of a generalhemostatic agent comprising administering a therapeutically effectiveamount of the chimeric protein prepared by the present methods.

The present invention is also related to methods of reducingimmunogenicity of FVIII or inducing less immunogenicity against FVIIIcomprising administering an effective amount of the chimeric proteinsdescribed herein, or the polynucleotides encoding the same.

In one embodiment, the subject in need of a general hemostatic agent isundergoing, or is about to undergo, surgery. The chimeric protein of theinvention can be administered prior to, during, or after surgery as aprophylactic regimen. The chimeric protein of the invention can beadministered prior to, during, or after surgery to control an acutebleeding episode.

The chimeric protein of the invention can be used to treat a subjecthaving an acute bleeding episode who does not have a hemostaticdisorder. The acute bleeding episode can result from severe trauma,e.g., surgery, an automobile accident, wound, laceration gun shot, orany other traumatic event resulting in uncontrolled bleeding. Nonlimiting examples of bleeding episodes include a bleeding coagulationdisorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhageinto muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinalbleeding, intracranial hemorrhage, intra-abdominal hemorrhage,intrathoracic hemorrhage, bone fracture, central nervous systembleeding, bleeding in the retropharyngeal space, bleeding in theretroperitoneal space, bleeding in the illiopsoas sheath, and anycombinations thereof.

In prophylactic applications, one or more compositions containing thechimeric protein of the invention or a cocktail thereof are administeredto a patient not already in the disease state to enhance the patient'sresistance or reduce symptoms associated with a disease or disorder.Such an amount is defined to be a “prophylactic effective dose.” Intherapeutic applications, a relatively high dosage (e.g., from about 1to 400 mg/kg of polypeptide per dose, with dosages of from 5 to 25 mgbeing more commonly used for radioimmuno conjugates and higher doses forcytotoxin-drug modified polypeptides) at relatively short intervals issometimes required until progression of the disease is reduced orterminated, and until the patient shows partial or complete ameliorationof symptoms of disease. Thereafter, the patient can be administered aprophylactic regime.

In some embodiments, a chimeric protein or a composition of theinvention is used for on-demand treatment, which includes treatment fora bleeding episode, hemarthrosis, muscle bleed, oral bleed, hemorrhage,hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis (headtrauma), gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, or bleeding in the illiopsoassheath. The subject may be in need of surgical prophylaxis,pen-operative management, or treatment for surgery. Such surgeriesinclude, e.g., minor surgery, major surgery, tooth extraction,tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement,craniotomy, osteosynthesis, trauma surgery, intracranial surgery,intra-abdominal surgery, intrathoracic surgery, or joint replacementsurgery.

In one embodiment, the chimeric protein of the present invention isadministered intravenously, subcutaneously, intramuscularly, or via anymucosal surface, e.g., orally, sublingually, buccally, nasally,rectally, vaginally or via pulmonary route. The chimeric proteincomprising a VWF fragment and a FVIII protein of the present inventioncan be implanted within or linked to a biopolymer solid support thatallows for the slow release of the chimeric protein to the site ofbleeding or implanted into bandage/dressing. The dose of the chimericprotein will vary depending on the subject and upon the particular routeof administration used. Dosages can range from 0.1 to 100,000 μg/kg bodyweight. In one embodiment, the dosing range is 0.1-1,000 μg/kg. Inanother embodiment, the dosing range is 0.1-500 μg/kg. The protein canbe administered continuously or at specific timed intervals. In vitroassays may be employed to determine optimal dose ranges and/or schedulesfor administration. In vitro assays that measure clotting factoractivity are known in the art, e.g., STA-CLOT VIIa-rTF clotting assay orROTEM clotting assay. Additionally, effective doses may be extrapolatedfrom dose-response curves obtained from animal models, e.g., ahemophiliac dog (Mount et al. 2002, Blood 99(8):2670).

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention. All patents, publications, andarticles referred to herein are expressly and specifically incorporatedherein by reference.

EXAMPLES

Throughout the examples, the following materials and methods were usedunless otherwise stated.

Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, biophysics,molecular biology, recombinant DNA technology, immunology (especially,e.g., antibody technology), and standard techniques in electrophoresis.See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: ColdSpring Harbor Laboratory Press (1989); Antibody Engineering Protocols(Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996);Antibody Engineering: A Practical Approach (Practical Approach Series,169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual,Harlow et al., CS. H. L. Press, Pub. (1999); and Current Protocols inMolecular Biology, eds. Ausubel et al., John Wiley & Sons (1992).

Example 1: Cloning Different VWF Domains (FIG. 1)

(a) Cloning of pSYN-VWF-002

pSYN-VWF-002 contains nucleotide sequences encoding a VWF fragment,which are amino acids 1-477 of SEQ ID NO: 100. [VWF-D′D3 proteinsequence] Amino acid numbering represents the mature VWF sequencewithout propeptide and corresponds to amino acids 764-1240 of SEQ ID NO:2. pSYN-VWF-002 construct has the FVIII signal peptide at N-terminus,which allows proper secretion of the synthesized protein and followed bya 6×His tag at C-terminus, which is used for protein purification. Itwas synthesized by using following primer combinations:

ESC48-Fwd-VWF-D′D3 with VIII signal and BsiW1 site (SEQ ID NO: 90)TCGCGACGTACGGCCGCCACCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGCCTATCCTGTCGGCCCCCCATGESC51-Rev-VWF D′D3 (1-477 amino acid) with 6His and Not 1 site(SEQ ID NO: 91) TGACCTCGAGCGGCCGCTCAGTGGTGATGGTGATGATGCGGCTCCTGGCAGGCTTCACAGGTGAGGTTGACAAC

A 50 μl PCR reaction was carried out with ESC 48/ESC 51 primercombinations and full length VWF plasmid as the template, using the 2step PCR amplification cycle: 94° C. 2 minutes; 21 cycles of (96° C. 30seconds, 68° C. 2 minute). The 1460 bp band was gel purified with a GelExtraction kit (Qiagen, Valencia, Calif.) and cloned into the BsiWI andNot1 restriction sites of pcDNA 4 to generate pSYN-VWF 002.

(b) Cloning of pSYN-VWF-010 and 013

pSYN-VWF-010 was constructed using pSYN-VWF-008 and pSYN-VWF-002.pSYN-VWF-008 contains the full-length VWF sequence in pcDNA 3.1 (aminoacids 1-2813 of SEQ ID NO: 2), it includes 763 amino acid propeptide(i.e., D1D2 domains) followed by remaining 2050 amino acids sequence ofmature VWF. The FVIII signal peptide in pSYN-VWF-002 was replaced withD1D2 domains from pSYN-VWF-008, the resulting construct is pSYN-VWF-010.pSYN-VWF-008 has a BamH1 site at Arg907 and Not1 site at the end ofcoding region (after stop codon). pSYN-VWF-008 and 002 were digestedwith BamH1 and Not1 restriction enzymes. Inserts from pSYN-VWF-002 (1026bp) were ligated into bamH1/Not1 digested pSYN-VWF-008 (8242 bp) toobtain pSYN-VWF-010 (D1D2D′D3: amino acid 1-1240 of SEQ ID NO: 2), a6×His tag was also added at the C-terminus. In transformed cellspSYN-VWF-010 is synthesized with propeptide but due to intracellularprocessing the secreted products do not contain any propeptide (D1D2).Protein from VWF-010 exists as dimer.

pSYN-VWF-010 was used to generate pSYN-VWF-013 which has two pointmutations at C336A and C379A corresponding to SEQ ID NO: 100 (amino acidnumbering represents mature VWF sequence without D1D2 domain-VWFsequence 2). These mutations are predicted to prevent dimerization ofVWF D′D3 domain.

(c) Cloning of pSYN-VWF-025 and pSYN-VWF-029

pSYN-VWF-025 contains wild type D1D2D′D3 sequences of full-length VWF inpLIVE vector, and pSYN-VWF-029 contains D1D2D′D3 sequence with C336A andC379A mutation. For cloning pSYN-VWF-025, the following primercombination was used:

ESC 89-fwd with Nhe1 site = (SEQ ID NO: 92)CTCACTATAGGGAGACCCAAGCTGGCTAGCCG ESC 91-rev with Sal1 = (SEQ ID NO: 93)CTGGATCCCGGGAGTCGACTCGTCAGTGGTGATGGTGATGATG

A 50 μl PCR reaction was carried out with ESC 89/ESC91 primercombinations and either pSYN-VWF 010 (for pSYN-VWF-025) or pSYN-VWF 013(for pSYN-VWF-029) plasmid as the template using the 3 step PCRamplification cycle: 94° C. 2 minutes; 21 cycles of (96° C.-30 seconds,55° C.-30 second, 68° C.-4 minutes). The expected sized band (˜3800 bp)was gel purified with a Gel Extraction kit (Qiagen, Valencia, Calif.)and cloned into the Nhe1 and Sal1 restriction sites of pLIVE-Mirusvector (Invitrogen, Carlsbad, Calif.) to generate pSYN-VWF 025 and 029.

(d) Cloning pSYN-VWF-031

pSYN-VWF-031 is a D1D2D′D3(C336A/C379A)-Fc construct which has a 48amino acid long thrombin cleavable linker (8× GGGGS (SEQ ID NO94)+thrombin site) in between the VWF D1D2D′D3(C336A/C379A) and the Fcsequences. To make this construct, VWF-Fc region was amplified fromconstruct pSYN-FVIII-064 (refer FVIII-VWF construct below).pSYN-FVIII-VWF was digested with Xba1 and Nhe1. Resulting insert regionof 4165 bp, containing the VWF fragment and Fc region was used as atemplate for amplifying the VWF and Fc region by primer combinations LW22/LW23.

LW 22-FWD-VWF-D′D3 with FVIII signal sequence and BsiW1 site(SEQ ID NO: 95) GCGCCGGCCGTACGATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGCCTATCCTGTCGGCCCCCCATGLW 23-Rev-Fc with stop codon and Not1 site (SEQ ID NO: 96)TCATCAATGTATCTTATCATGTCTGAATTCGCGGCCGCTCATTTACC

The PCR product obtained from LW22/LW23 amplification (˜2300 bp) wascloned in BsiW1/Not1 digested pSYN-VWF-002 to obtain pSYN-VWF-014intermediate. pSYN-VWF-014 contains FVIII signal peptide-D′D3-20 aminoacid thrombin cleavable linker followed by the Fc region.

To generate the D1D2D′D3-Fc construct, the D1D2D′D3 region was amplifiedfrom pSYN-VWF-013 using primer combination LW24/LW27 by standard PCRmethod.

LW24-Fwd-VWF D1D2D′D3 cloning oligo with BsiW1 site (SEQ ID NO: 97)GCGCCGGCCGTACGATGATTCCTGCCAGATTTGCCGGGGTGLW27-Rev-VWF D′D3 oligo with EcoRV (SEQ ID NO: 98)CCACCGCCAGATATCGGCTCCTGGCAGGCTTCACAGGTGAG

The PCR product obtained from LW22/LW23 amplification (˜3750 bp) wascloned in BsiW1/EcoRV digested pSYN-VWF-014 to obtain pSYN-VWF-015intermediate. The linker length between the VWF fragment and Fc regionwas changed to obtain pSYN-VWF-031.

VWF-D1D2D′D3 protein sequence 1 (SEQ ID NO: 99)    1MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM   51YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG  101TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL  151SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC  201ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC  251EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME  301YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC  351VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD  401NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG  451LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM  501DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG  551NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS  601PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL  651NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD  701CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD  751AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM  801SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV  851CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS  901NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE  951THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCACF 1101CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY ECEWRYNSCA 1151PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP* VWF-D′D3 protein sequence 2(SEQ ID NO: 100)    1SLSCRPPMVK LVCPADNLRA EGLECTKTCQ NYDLECMSMG CVSGCLCPPG   51MVRHENRCVA LERCPCFHQG KEYAPGETVK IGCNTCVCRD RKWNCTDHVC  101DATCSTIGMA HYLTFDGLKY LFPGECQYVL VQDYCGSNPG TFRILVGNKG  151CSHPSVKCKK RVTILVEGGE IELFDGEVNV KRPMKDETHF EVVESGRYII  201LLLGKALSVV WDRHLSISVV LKQTYQEKVC GLCGNFDGIQ NNDLTSSNLQ  251VEEDPVDFGN SWKVSSQCAD TRKVPLDSSP ATCHNNIMKQ TMVDSSCRIL  301TSDVFQDCNK LVDPEPYLDV CIYDTCSCES IGDCACFCDT IAAYAHVCAQ  351HGKVVTWRTA TLCPQSCEER NLRENGYECE WRYNSCAPAC QVTCQHPEPL  401ACPVQCVEGC HAHCPPGKIL DELLQTCVDP EDCPVCEVAG RRFASGKKVT  451LNPSDPEHCQ ICHCDVVNLT CEACQEP

Example 2: Effects of D′D3 and XTEN Fusion on FVIII Half-Life Extension

To evaluate D′D3 FVIII half-life extension potential on rFVIII-XTENfusion protein, a VWF D′D3 dimer was introduced into FVIII-VWF DKO miceby hydrodynamic injection of its corresponding DNA construct VWF-025(Example 1). After D′D3 has reached the steady state expression (day5post injection), a single dose of rFVIII-XTEN was administered by IVinjection at 200 IU/kg dose. Blood samples were collected up to 120 hrspost rFVIII-XTEN dosing. Plasma FVIII activity was analyzed by a FVIIIchromogenic assay. The D′D3 expression level was measured by VWF ELISA,and rFVIIIFc PK profile was analyzed using WinNonlin program.

The study results were shown in FIG. 2, and the PK parameter ofrFVIII-XTEN with/without D′D3 in circulation was listed in Table 16. TheD′D3 dimer further extended rFIII-XTEN t_(1/2) from 3.4 hr to 17.8 hr, a5 fold increase. In addition to half-life, 5 fold of increase on MRT,3.6 fold increases on AUC, 3.8 fold decreases on clearance were alsoobserved.

We have observed a synergistic effect of D′D3 fragment and XTENtechnology, a serial of FVIII/VWF/XTEN constructs will be evaluated fortheir FVIII half-life extension potential in Hemophilic animals.

TABLE 16 rFVIII-XTEN PK parameter with/without D′D3 in blood circulation5 min Cl AUC_D Recovery t_(1/2) MRT (mL/ Vss (hr*kg*mIU/ Treatment (%)(hr) (hr) hr/kg) (mL/kg) mL/mIU) rFVIIIXTEN 80 17.8 19.3 3.5 67.4 0.29VWF-025 rFVIIIXTEN 74 3.4 3.8 13.1 63.68 0.08 Improvement 1.1 5.2 5.13.8 0.9 3.6 fold

Protein Purification of FVIII-XTEN

An AE288 XTEN was inserted at the C-terminus of BDD-FVIII for thisstudy. To purify this protein, a tangential flow filtration (TFF) stepwas used first to buffer exchange the conditioned media. Products in thefiltrate were then captured using a strong anion exchangechromatography, and then further purified using affinity chromatography.Purity of the molecule was acceptable by HPLC-SEC and was furtherconfirmed by western blotting. The specific activity of the molecule wascomparable to B-domain deleted FVIII, as measured by aPTT assay andELISA.

FVIII Chromogenic Assay

The FVIII activity was measured using the COATEST SP FVIII kit fromDiaPharma (lot #N089019) and all incubations were performed on a 37° C.plate heater with shaking.

The range of rFVIII standard was from 100 mIU/mL to 0.78 mIU/mL. Apooled normal human plasma assay control and plasma samples (dilutedwith 1× Coatest buffer) were added into Immulon 2HB 96-well plates induplicate (25 μL/well). Freshly prepared IXa/FX/Phospholipid mix (50μL), 25 μL of 25 mM CaCl2, and 50 μL of FXa substrate were addedsequentially into each well with 5 minutes incubation between eachaddition. After incubating with the substrate, 25 μL of 20% Acetic Acidwas added to terminate the color reaction, and the absorbance of OD405was measured with a SpectraMAX plus (Molecular Devices) instrument. Datawere analyzed with SoftMax Pro software (version 5.2). The Lowest Levelof Quantification (LLOQ) is 7.8 mIU/mL.

VWF ELISA:

Goat anti-human VWF antibody (Affinity purified, affinity biological,GAVWF-AP) was used as the capture antibody at 0.5 ug/well and VWF-EIA-D(Affinity Biologicals, VWF-EIA-D, 1:100 dilution) was used as thedetecting antibody for the VWF ELISA. ELISA assay was performedfollowing the standard ELISA procedure, TMB was used as the HRPsubstrate, PBST/1.5% BSA/0.5M NaCl buffer was used as blocking andbinding buffer. The assay standard range is 100 ng to 0.78 ng, andassay's lowest limit of quantification (LLOQ) is 7.8 ng/mL.

Example 3: Plasmid Construction of XTEN Containing FVIII/VWF Constructs

(a) Cloning of pSYN-FVIII-161 (FIG. 3)

The FVIII-161 plasmid comprises a single chain Fc (scFc) scaffold withenzyme cleavage sites which are processed during synthesis in a cell.The construct has a FVIII binding domain of full-length VWF (D′D3).

Plasmid (pSYN-FVIII-161) was designed for the expression FVIII-Fc andVWF-Fc heterodimer, where the D′D3 domains to bind FVIII and preventsFVIII interaction with phospholipids and activated protein C. Proteinfrom pSYN-FVIII-161 is expressed in the cell as a single polypeptidewhere the C-terminus of the FVIII-Fc subunit is linked to the N-terminusof the VWF D′D3-Fc subunit by a 6× (GGGGS) polypeptide linker (SEQ IDNO: 64). In addition, RRRRS (SEQ ID NO: 11) and RKRRKR (SEQ ID NO: 10)sequences were inserted at the 5′ and 3′ end of the polypeptide linker,respectively, for intracellular cleavage by proprotein convertasesfollowing the last Arg at each sequence. Hence, the cells can express adouble chain FVIII-Fc/D′D3-Fc heterodimer where the FVIII-Fc chain has aRRRRS sequence (SEQ ID NO: 11) at the C-terminus, but the remainder ofthe linker sequence has been removed. An AE288 XTEN fragment immediatelyfollowed by IS{5X(GGGGS)}LVPRGSGG (SEQ ID NO: 122) polypeptide (containsthrombin cleavage site) is introduced in between the VWF domains and theFc region to facilitate release of the VWF fragment from FVIII once theFVIII-VWF hetero-dimeric protein is activated by thrombin allowinginteraction of FVIII with other clotting factors.

pSYN-FVIII-161 (SEQ ID NO: 101). protein sequence (FVIII sequenceamino acid position 1-1457; underlined region represents Fc region; curvyunderline represents cleavable linker in between first Fc and VWF fragment;double underlined region represents VWF fragment; bold region representscleavable linker in between VWF fragment and Fc. 1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP 51PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY 101DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG 151GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE 201GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM 251HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH 301RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE 351EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT 401WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY 451TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT 501DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR 551YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE 601NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL 651HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS 701MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL 751SKNNAIEPRS FSQNPPVLKR HQREITRTTL QSDQEEIDYD DTISVEMKKE 801DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 851SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 901RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 951TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1001FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI 1051MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1101YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1151GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1201LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1251FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1301MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1351PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1401QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1451CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1501VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 1551LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 1601SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 1651

1701

DNLRAEGLEC 1751 TKTCQNYDLE CMSMGCVSGC LCPPGMVRHE NRCVALERCP CFHQGKEYAP1801 GETVKIGCNT CVCRDRKWNC TDHVCDATCS TIGMAHYLTF DGLKYLFPGE 1851CQYVLVQDYC GSNPGTFRIL VGNKGCSHPS VKCKKRVTIL VEGGEIELFD 1901GEVNVKRPMK DETHFEVVES GRYIILLLGK ALSVVWDRHL SISVVLKQTY 1951QEKVCGLCGN FDGIQNNDLT SSNLQVEEDP VDFGNSWKVS SQCADTRKVP 2001LDSSPATCHN NIMKQTMVDS SCRILTSDVF QDCNKLVDPE PYLDVCIYDT 2051CSCESIGDCA AFCDTIAAYA HVCAQHGKVV TWRIATLCPQ SCEERNLREN 2101GYEAEWRYNS CAPACQVTCQ HPEPLACPVQ CVEGCHAHCP PGKILDELLQ 2151TCVDPEDCPV CEVAGRRFAS GKKVTLNPSD PEHCQICHCD VVNLTCEACQ 2201

GSEPATSGSE 2251 TPGTSESATP ESGPGTSTEP SEGSAPGSPA GSPTSTEEGT SESATPESGP2301 GSEPATSGSE TPGTSESATP ESGPGSPAGS PTSTEEGSPA GSPTSTEEGT 2351STEPSEGSAP GTSESATPES GPGTSESATP ESGPGTSESA TPESGPGSEP 2401ATSGSETPGS EPATSGSETP GSPAGSPTST EEGTSTEPSE GSAPGTSTEP 2451SEGSAPGSEP ATSGSETPGT SESATPESGP GTSTEPSEGS APDSGGGGSG 2501GGGSGGGGSG GGGSGGGGSL VPRGSGG DKT HTCPPCPAPE LLGGPSVFLF 2551PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE 2601EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP 2651REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT 2701TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL 2751 SPGK

(b) Cloning of pSYN-FVIII-168, 175, 172 and 174 (FIG. 4A-4D)

pSYN-FVIII-168, 172, 174 and 175 are derivatives of pSYN-FVIII-161.R1645A/R1648A mutations were introduced into pSYN-FVIII-161 to formpSYN-FVIII-168, which produces a SC-FVIII isoform, and an AE288 XTEN wasdirectly fused into the C-terminus of FVIII-HC for further half-lifeextension. To construct pSYN-FVIII-175, the D′D3 codon sequence wasremove form pSYN-FVIII-168 for evaluation of the effect of Fc and XTENtechnology on FVIII half-life extension.

To construct pSYN-FVIII-172, the AE288 XTEN fragment was directly fusedinto the C-terminus of FVIII-HC for further half-life extension, and theD′D3 codon sequence was removed from pSYN-FVIII-172 to formpSYN-FVIII-174 for evaluation of the effect of Fc and XTEN technology onFVIII half-life extension.

(c) Cloning of pSYN-FVIII-170 (FIG. 4E)

pSYN-FVIII-170 was constructed to evaluate the effect of XTEN and D′D3fragment on FVIII half-life extension. The codon sequence VWF-D1D2D′D3fragment and BDD-FVIII were introduced into the 5′ and 3′ end ofexpression casket, an AE288 XTEN codon sequence which followed by a 35aa thrombin cleavable linker was used to connect the VWF and FVIIImolecule. After intra cellular processing, the secreted proteincomprises a polypeptide contains the D′D3 fragment of mature VWFmolecule which is linked to the N-terminus of mature BDD-FVIII by anAE288 XTEN/35 aa thrombin cleavable linker.

pSYN-FVIII-170 protein sequence (SEQ ID NO: 102)    1SLSCRPPMVK LVCPADNLRA EGLECTKTCQ NYDLECMSMG CVSGCLCPPG   51MVRHENRCVA LERCPCFHQG KEYAPGETVK IGCNTCVCRD RKWNCTDHVC  101DATCSTIGMA HYLTFDGLKY LFPGECQYVL VQDYCGSNPG TFRILVGNKG  151CSHPSVKCKK RVTILVEGGE IELFDGEVNV KRPMKDETHF EVVESGRYII  201LLLGKALSVV WDRHLSISVV LKQTYQEKVC GLCGNFDGIQ NNDLTSSNLQ  251VEEDPVDFGN SWKVSSQCAD TRKVPLDSSP ATCHNNIMKQ TMVDSSCRIL  301TSDVFQDCNK LVDPEPYLDV CIYDTCSCES IGDCAAFCDT IAAYAHVCAQ  351HGKVVTWRTA TLCPQSCEER NLRENGYEAE WRYNSCAPAC QVTCQHPEPL  401ACPVQCVEGC HAHCPPGKIL DELLQTCVDP EDCPVCEVAG RRFASGKKVT  451LNPSDPEHCQ ICHCDVVNLT CEACQEPISG TSESATPESG PGSEPATSGS  501ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG TSTEPSEGSA  551PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG  601SPAGSPTSTE EGSPAGSPTS TEEGTSTEPS EGSAPGTSES ATPESGPGTS  651ESATPESGPG TSESATPESG PGSEPATSGS ETPGSEPATS GSETPGSPAG  701SPTSTEEGTS TEPSEGSAPG TSTEPSEGSA PGSEPATSGS ETPGTSESAT  751PESGPGTSTE PSEGSAPDSG GGGSGGGGSG GGGSGGGGSG GGGSLVPRGS  801GGASATRRYY LGAVELSWDY MQSDLGELPV DARFPPRVPK SFPFNTSVVY  851KKTLFVEFTD HLFNIAKPRP PWMGLLGPTI QAEVYDTVVI TLKNMASHPV  901SLHAVGVSYW KASEGAEYDD QTSQREKEDD KVFPGGSHTY VWQVLKENGP  951MASDPLCLTY SYLSHVDLVK DLNSGLIGAL LVCREGSLAK EKTQTLHKFI 1001LLFAVFDEGK SWHSETKNSL MQDRDAASAR AWPKMHTVNG YVNRSLPGLI 1051GCHRKSVYWH VIGMGTTPEV HSIFLEGHTF LVRNHRQASL EISPITFLTA 1101QTLLMDLGQF LLFCHISSHQ HDGMEAYVKV DSCPEEPQLR MKNNEEAEDY 1151DDDLTDSEMD VVRFDDDNSP SFIQIRSVAK KHPKTWVHYI AAEEEDWDYA 1201PLVLAPDDRS YKSQYLNNGP QRIGRKYKKV RFMAYTDETF KTREAIQHES 1251GILGPLLYGE VGDTLLIIFK NQASRPYNIY PHGITDVRPL YSRRLPKGVK 1301HLKDFPILPG EIFKYKWTVT VEDGPTKSDP RCLTRYYSSF VNMERDLASG 1351LIGPLLICYK ESVDQRGNQI MSDKRNVILF SVFDENRSWY LTENIQRFLP 1401NPAGVQLEDP EFQASNIMHS INGYVFDSLQ LSVCLHEVAY WYILSIGAQT 1451DFLSVFFSGY TFKHKMVYED TLTLFPFSGE TVFMSMENPG LWILGCHNSD 1501FRNRGMTALL KVSSCDKNTG DYYEDSYEDI SAYLLSKNNA IEPRSFSQNP 1551PVLKRHQREI TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS 1601FQKKTRHYFI AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD 1651GSFTQPLYRG ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI 1701SYEEDQRQGA EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD 1751VDLEKDVHSG LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY 1801FTENMERNCR APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI 1851RWYLLSMGSN ENIHSIHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP 1901SKAGIWRVEC LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS 1951GQYGQWAPKL ARLHYSGSIN AWSTKEPFSW IKVDLLAPMI IHGIKTQGAR 2001QKFSSLYISQ FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF 2051NPPIIARYIR LHPTHYSIRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI 2101TASSYFTNMF ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV 2151TGVTTQGVKS LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF 2201TPVVNSLDPP LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LY

Example 4: Hydrodynamic Injection of XTEN Containing FVIIIF/VWFConstructs in FVIII and VWF Deficient Mice

The XTEN containing DNA constructs in FIGS. 3 and 4 have combined 2-3half-life extension elements together. To evaluate their FVIII half-lifeextension potential, a selective group of DNA constructs in FIG. 3 andFIG. 4 were introduced into FVIII/VWF double knockout (DKO) mice byHydrodynamic injection (HDI) at 100 ug/mouse dose. Blood samples werethen collected by retro orbital blood collection at 24 hr post HDI. Thepost HDI plasma FVIII activity was analyzed by FVIII chromogenic assay,and results were listed in Table 17 and FIG. 5. Compared to wild typeBDD-FVIII, all XTEN containing DNA constructs yield significantly higherFVIII plasma activity at 24 hr post HDI, indicating the correspondingmolecules had significant longer circulating protein half-life thanBDD-FVIII. The application of the combination of those half-lifeextending elements was further evaluated in Hemophilic animals.

TABLE 17 FVIII plasma activity 24 hr post HDI in FVIII/VWF DKO mice DNABDD- FVIII- FVIII- BDD- FVIII- Construct FVIII FVIII-161 168 172 FVIII170 DNA Dose 100 100 100 100 50 50 (μg/mouse) FVIII 219 ± 2446 ± 2209 ±1671 ± 197 ± 399 ± Activity 72 1012 609 223 21 30 (mU/mL)

Hydrodynamic Injection:

Hydrodynamic Injection is an efficient and safe non-viral gene deliverymethod to the liver in small animals, such as mice and rats. It wasoriginally described as a rapid injection of a naked plasmid DNA/salinesolution free of endotoxin at a tenth volume of the animal's body weightin about 5-7 seconds. The naked plasmid DNA contains the gene ofinterest and the liver produced in a tenth volume of the animal's bodyweight. The targeted protein is produced in the liver from the injectedDNA and can be detected within 24 hours post-injection. Plasma sampleswere then collected to study the therapeutic property of the expressedprotein.

For all the hydrodynamic injections that were performed herein, 2 ml ofplasmid DNA in 0.9% sterile saline solution was delivered viaintravenous tail vein injection within about 4-7 seconds to miceweighing 20-35 grams. The mice were closely monitored for the firstcouple of hours until the normal activity resumed. After the bloodsamples were collected via retro orbital blood collection, plasmasamples were then obtained and stored at −80° C. for further analysis.

Example 5: Plasmid Construction of Co-Transfection System forFVIIIFc-VWF Heterodimer Contain XTEN Insertions (FIG. 6)

To increase the protein production yield, two co-transfection systemswere generated for protein production, which contains three DNAconstructs. The first DNA construct encoded a FVIII-Fc fusion protein inwhich a AE288 XTEN fragment was directly fuse to the C-terminus of theFVIII heavy chain and followed by either a wild type FVIII light chainfragment (pSYN-FVIII-173, FIG. 6B) or a FVIII light chain fragment withR1645A/R1648A mutations (pSYN-FVIII-169, FIG. 6A), the FVIII light chainwas then directly fused to a single Fc fragment. The second DNAconstruct is pSYN-VWF-031 which encoding a D′D3-Fc fusion protein(Example 1). HEK293F cells were transfected with the two plasmid alongwith a third plasmid (PC5) at 80:15:5 ratio. The synthesized proteinswere secreted as FVIII (XTEN) Fc/D′D3Fc heterodimer and D′D3Fc dimer andthe FVIII (XTEN) Fc/D′D3Fc heterodimer was separated from the D′D3Fcdimer by protein purification.

pSYN-FVIII-169 mature Protein sequence (SEQ ID NO: 103):    1ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSVVYKKTL   51FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTVVITLKN MASHPVSLHA  101VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD  151PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA  201VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR  251KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL  301MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL  351TDSEMDVVRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL  401APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG  451PLLYGEVGDT LLIIFKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD  501FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RDLASGLIGP  551LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN IQRFLPNPAG  601VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS  651VFFSGYTFKH KMVYEDTLIL FPFSGETVFM SMENPGLWIL GCHNSDFRNR  701GMTALLKVSS CDKNTGDYYE DSYEDISAYL LSKNNAIEPR SFSQNGAPGT  751SESATPESGP GSEPATSGSE TPGTSESATP ESGPGSEPAT SGSETPGTSE  801SATPESGPGT STEPSEGSAP GSPAGSPTST EEGTSESATP ESGPGSEPAT  851SGSETPGTSE SATPESGPGS PAGSPTSTEE GSPAGSPTST EEGTSTEPSE  901GSAPGTSESA TPESGPGTSE SATPESGPGT SESATPESGP GSEPATSGSE  951TPGSEPATSG SETPGSPAGS PTSTEEGTST EPSEGSAPGT STEPSEGSAP 1001GSEPATSGSE TPGTSESATP ESGPGTSTEP SEGSAPASSP PVLKRHQAEI 1051TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS FQKKTRHYFI 1101AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD GSFTQPLYRG 1151ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI SYEEDQRQGA 1201EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD VDLEKDVHSG 1251LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY FTENMERNCR 1301APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI RWYLLSMGSN 1351ENIHSIHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP SKAGIWRVEC 1401LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS GQYGQWAPKL 1451ARLHYSGSIN AWSTKEPFSW IKVDLLAPMI IHGIKTQGAR QKFSSLYISQ 1501FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF NPPIIARYIR 1551LHPTHYSIRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI TASSYFTNMF 1601ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV TGVTTQGVKS 1651LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF TPVVNSLDPP 1701LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LYDKTHTCPP CPAPELLGGP 1751SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK 1801TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK 1851AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE 1901NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ 1951 KSLSLSPGKpSYN-FVIII-173 mature Protein sequencing (SEQ ID NO: 104):    1ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSVVYKKTL   51FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTVVITLKN MASHPVSLHA  101VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD  151PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA  201VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR  251KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL  301MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL  351TDSEMDVVRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL  401APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG  451PLLYGEVGDT LLIIFKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD  501FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RDLASGLIGP  551LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN IQRFLPNPAG  601VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS  651VFFSGYTFKH KMVYEDTLIL FPFSGETVFM SMENPGLWIL GCHNSDFRNR  701GMTALLKVSS CDKNTGDYYE DSYEDISAYL LSKNNAIEPR SFSQNGAPGT  751SESATPESGP GSEPATSGSE TPGTSESATP ESGPGSEPAT SGSETPGTSE  801SATPESGPGT STEPSEGSAP GSPAGSPTST EEGTSESATP ESGPGSEPAT  851SGSETPGTSE SATPESGPGS PAGSPTSTEE GSPAGSPTST EEGTSTEPSE  901GSAPGTSESA TPESGPGTSE SATPESGPGT SESATPESGP GSEPATSGSE  951TPGSEPATSG SETPGSPAGS PTSTEEGTST EPSEGSAPGT STEPSEGSAP 1001GSEPATSGSE TPGTSESATP ESGPGTSTEP SEGSAPASSP PVLKRHQREI 1051TRTTLQSDQE EIDYDDTISV EMKKEDFDIY DEDENQSPRS FQKKTRHYFI 1101AAVERLWDYG MSSSPHVLRN RAQSGSVPQF KKVVFQEFTD GSFTQPLYRG 1151ELNEHLGLLG PYIRAEVEDN IMVTFRNQAS RPYSFYSSLI SYEEDQRQGA 1201EPRKNFVKPN ETKTYFWKVQ HHMAPTKDEF DCKAWAYFSD VDLEKDVHSG 1251LIGPLLVCHT NTLNPAHGRQ VTVQEFALFF TIFDETKSWY FTENMERNCR 1301APCNIQMEDP TFKENYRFHA INGYIMDTLP GLVMAQDQRI RWYLLSMGSN 1351ENIHSIHFSG HVFTVRKKEE YKMALYNLYP GVFETVEMLP SKAGIWRVEC 1401LIGEHLHAGM STLFLVYSNK CQTPLGMASG HIRDFQITAS GQYGQWAPKL 1451ARLHYSGSIN AWSTKEPFSW IKVDLLAPMI IHGIKTQGAR QKFSSLYISQ 1501FIIMYSLDGK KWQTYRGNST GTLMVFFGNV DSSGIKHNIF NPPIIARYIR 1551LHPTHYSIRS TLRMELMGCD LNSCSMPLGM ESKAISDAQI TASSYFTNMF 1601ATWSPSKARL HLQGRSNAWR PQVNNPKEWL QVDFQKTMKV TGVTTQGVKS 1651LLTSMYVKEF LISSSQDGHQ WTLFFQNGKV KVFQGNQDSF TPVVNSLDPP 1701LLTRYLRIHP QSWVHQIALR MEVLGCEAQD LYDKTHTCPP CPAPELLGGP 1751SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK 1801TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK 1851AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE 1901NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ 1951 KSLSLSPGK

Example 6. Protein Purification for FVIII-169/VWF-031 andFVIII-173/VWF-031

A tangential flow filtration (TFF) step was used to buffer exchange theclarified conditioned media. The FVIII-169NWF-031 or FVIII-173NWF-031heterodimer was then purified using a two-step chromatography process. Aweak anion exchange resin was used, followed by affinity chromatography.The final purified product had acceptable purity by SEC-HPLC. Thespecific activity was compatible to B-domain deleted FVIII, as measuredby FVIII chromogenic assay and A280 concentration. Purity and thepresence of each moiety of this molecule were confirmed by SDS-PAGE andwestern blotting.

Example 7. Evaluation the VWF Binding Ability of FVIII-169/VWF-031 byOctet Assay

The VWF binding ability of FVIII-169/VWF-031 was obtained by Bio-LayerInterferometry (BLI) based measurements (Octet assay) at 25° C. with aForteBio Octet 384 instrument, using Tris binding buffer (50 mM Tris, pH7.2, 150 mM NaCl, 5 mM CaCl₂)). The Octet assay for determining FVIIIbinding was based on the hydrophobic immobilization of Human vonWillebrand Factor (Haematologic Technologies Catalog No. HCVWF-0191)onto the APS Biosensor, then followed by the binding of 1.0% BovineSerum Albumin (Jackson ImmunoResearch Catalog No. 001-000-161). Briefly,hvWF (20 μg/mL) was diluted in Tris buffer and loaded across APSBiosensors for 600 sec, yielding approximately 3.0-3.5 nm binding on thereaction probes. Control APS probes were loaded with 1.0% BSA in theabsence of hvWF for reference subtraction. After loading, all probeswere incubated in Tris buffer for 300 sec to establish a new baseline.Subsequently, biosensor probes were incubated in solutions of FVIII-XTEN169 or FVIIIFc Drug Substance (0, 0.6, 2, 6, 20, 60, 200, 600 IU/mL) for5 min at room temperature, followed by a 5 min dissociation step. Usingthe Octet data analysis software, the binding response (nm) was derivedfrom the subtracted data (Reaction probe minus Reference probe). Nobinding to immobilized VWF was detected for FVIII-169/VWF-031 (FIG. 7),indicating a complete shielding of FVIII from full length VWF moleculeby the D′D3 fragment.

Example 8. FVIII-169/VWF-031 PK in HemA and FVIII/VWF DKO Mice

The PK profile of FVIII-169/VWF-031 was tested in HemA and FVIII/VWF DKOmice to evaluate the ability of the D′D3 fragment to shield the FVIIImoiety from the endogenous VWF. HemA or FVIII/VWF DKO mice were treatedwith a single intravenous dose of FVIII-169/VWF-031 at 200 IU/kg, plasmasamples were then collected at 5 min, 8 hr, 24 hr, 48 hr and 72 hourspost dosing. The FVIII activity of plasma sample was tested by FVIIIchromogenic assay, and half-life of FVIII-169/VWF-031 was calculatedusing WinNonlin program.

Complete inhibition of the constructs' binding to immobilized VWF wasdemonstrated by biolayer interferometry (FIG. 7) for FVIII-169/VWF-031.This indicates the D′D3 fragment in the molecule had successfullyblocked the FVIII binding to native VWF molecules, therefor similarhalf-life of FVIII-169/VWF-031 was predicted in the two different mousestrains. As shown in FIG. 8A and Table 18, as expected,FVIII-169/VWF-031 had similar PK profile in both HemA and FVIII/VWF DKOmice, which has demonstrated that the half-life of FVIIIFc/VWFheterodimer is independent from the half-life of endogenous VWF. Theseparation of the FVIIIFc/VWF heterodimer half-life from the endogenousVWF half-life, eliminated the FVIII extension ceiling and opened thepossibility of further extending FVIII half-life beyond the 2 foldhalf-life limit imposed by endogenous VWF.

TABLE 18 FVIII-169/VWF-031 PK in HemA and FVIII/VWF DKO mice Cl AUCMouse Recovery t_(1/2) MRT (mL/hr/ Vss (hr*kg*mIU/ Strain (%) (hr) (hr)kg) (mL/kg) mL/mIU) FVIII/VWF 69 17.94 20.1  4.06 81.69 0.2461 DKO HemA83 16.65 18.44 3.57 85.72 0.28 

The FVIII protecting ability of the XTEN insertion and D′D3 fragment wasevaluated by comparing the half-life of FVIII-169/VWF-031 withFVIII-169/Fc and FVIIIFc in FVIII/VWF DKO mice. After a single IVadministration, blood samples were collected at 5 min, 8 hr, 24 hr, 48hr and 72 hr for FVIII-169/VWF-031, 5 min, 8 hr, 24 hr, 32 hr, 48 hr forFVIII-169/Fc and at 5 min, 1, 2, 4, 6 and 8 hrs for FVIIIFc. The FVIIIactivity of plasma sample was tested by FVIII chromogenic assay, andhalf-life of FVIII-155/VWF-031 was calculated using WinNonlin program.

The study results were summarized in FIG. 8B and Table 19, rFVIIIFc hasa 1.6 hr half-life in DKO mice due to the loss of VWF protection. Whenan XTEN insertion was introduced into the FVIIIFc molecule, theresulting FVIII-169/Fc molecule has a 7 hr half-life, a 4 fold half-lifeextension by the XTEN insertion. Finally, when D′D3 fragment wasincorporated into the molecule to form FVIII-169/VWF-031, a 17 hrhalf-life was observed, another 2.5 fold further increase by the D′D3fragment. In addition of the half-life improvement, improved Meanresidency time (MRT), Clearance (C1) and AUC were also observed as shownin Table 19.

FVIII-169/VWF-031 has achieved 17-18 hr t_(1/2) in both HemA andFVIII/VWF DKO mice, which is the upper limit of the t_(1/2) extensionceiling that imposed by VWF clearance. More t_(1/2) extension elementscan be further incorporated into this molecule, such as a second XTENinsertion within FVIII. The synergistic effect of D′D3 fragment and XTENinsertions provided the possibility of the complete protection for FVIIIfrom its clearance pathway, a final breakthrough of the 2 fold FVIIIt_(1/2) extension limit might be achieved by the FVIIIFc/XTEN/VWFvariants.

TABLE 19 FVIII-169/VWF-031 PK in FVIII/VWF DKO mice AUC/D Re- Cl (hr*kg*Mouse covery t_(1/2) MRT (mL/hr/ Vss mIU/mL/ Strain Treatment (%) (hr)(hr) kg) (mL/kg) mIU) FVIII/ rFVIIIFc 35  1.6  2.1 57.7 120.2 0.0173 VWFrFVIII- 77  7.0  6.2  6.4  39.2 0.1573 DKO 169/Fc rFVIII- 69 17.9 20.1 4.1  81.7 0.2461 169/ VWF-031

Example 9: FVIII-XTEN Variants Cell Media Concentrate PK in D′D3Expressing FVIII/VWF DKO Mice

The ability of D′D3 fragment to extend the t_(1/2) of FVII-XTEN wasevaluated in the D′D3 expressing FVIII/VWF DKO mouse model (described inexample 2). In this study, instead of using VWF-025 to introduce theD′D3 dimer into the circulation, VWF-029 construct was used to introducethe D′D3 monomer into the circulation. To prepare FVIII-XTEN variantsprotein, a small scale (50-100 mL) transient transfection culture mediawas prepared, at day 4 post transfection, cell culture was harvested andconcentrated to reach 10-20 IU/mL of FVIII activity range which issuitable for PK study. The concentrated cell media were then used forstandard PK study in FVIII/VWF DKO mice with or without D′D3 in thecirculation.

Total of 6 FVIII-XTEN variants that contains 1-3 XTEN insertions weretested in the system, their t_(1/2) were summarized in Table 20 and datafrom representative variants were plotted in FIG. 9A.

Longer half-life was observed for all the FVIII-XTEN variants with thepresents of D′D3 fragment in the circulation (Table 20), whichdemonstrated the D′D3 protection for FVIII-XTEN from its clearancepathways. Furthermore, when compared to its 14 hr half-life in HemAmice, LSD0055.021 has a 20.4 hr t_(1/2) in D′D3 expressing DKO mice(FIG. 9B, Table 20), indicates the final breakthrough of the 2 foldhalf-life extension ceiling for FVIII molecules. By further modify theFVIII(XTEN)/VWF molecule, we could potentially achieve even longer FVIIIt_(1/2), and provide HemA patients a FVIII protein that only requiresonce weekly or less frequent dosing regimen.

TABLE 20 FVIII-XTEN t_(1/2) in D′D3 expressing FVIII/VWF DKO mice # oft_(1/2) (hr) t_(1/2) XTEN t_(1/2) (hr) pLIVE- (hr) FVIII-XTEN inser-Insertion XTEN DKO D′D3/ HemA ID tions sites size mice DKO mice micepSD-0013 1 CT 144 3.3 7.9 LSD0003.009 2 B*/CT 144/288 9.7 16.4LSD0038.015 2 1656/26 144/144 7.8 17.2 LSD0049.002 3 18/B*/ 144/144/12.6 17.5 CT 288 LSD0051.002 3 403/B*/ 144/144/ 11.1 19.9 CT 288LSD0055.021 3 1900/B*/ 144/144/ 16 20.4 14 CT 288 *B indicates an XTENsequence (e.g., 144) is inserted immediately downstream of amino acidresidue 745 corresponding to mature FVIII sequence.

Example 10: Stability of VWF- and XTEN-Containing FVIII Variants inFVIII/VWF Double Knockout (DKO) Plasma

Plasma stability of rFVIIIFc protein variants was tested in FVIII/VWFdouble knockout (DKO) mouse plasma. For the stability assay, HEK293cells were co-transfected with plasmids directing the expression ofrFVIIIFc or FVIII-169 (rFVIIIFc with 288 AE XTEN inserted at theB-domain junction) and plasmids directing the expression of eitherIgG-Fc or VWF-031 (VWF D′D3 region fused to IgG-Fc). At day fourpost-transfection, cell culture media was harvested and concentrated to30 IU/mL based on FVIII chromogenic activity. Concentrated cell culturemedium was then added into DKO mouse plasma to yield a FVIII activity of5 IU/mL and incubated at 37° C. Aliquots were collected at differenttime points for activity measurement by chromogenic assay. Activity ateach time point was measured in duplicate, and the average activity wasplotted as a function of time. The activity of FVIIIFc, a dual chain(dc) FVIII molecule in which heavy and light chains are held together bynon-covalent interaction, decreases with time in DKO mouse plasma (FIG.10). The activity of FVIII-169:Fc, which contains a 288 AE XTENinsertion at the B-domain junction, decays at a reduced rate relative torFVIIIFc, indicating that enhanced stability is conferred by the XTENinsertion. Given that VWF has been proposed to enhance the stability ofFVIII in vivo, we evaluated the plasma stability of FVIII-169:VWF-031.This heterodimeric molecule, in which the FVIII element and the VWF D′D3element are fused to respective hemi-domains of Fc, exhibited additionalplasma stability relative to FVIII-169:Fc, indicating that the VWF D′D3domain and XTEN have a synergistic effect on the plasma stability ofrFVIIIFc. Example 11: The effect on FVIII half-life of Fc fusion, XTENinsertion and the D′D3 fragment of VWF.

To assess the effect of Fc fusion, XTEN insertion and D′D3 fragment ofVWF on the half-life of FVIII, the pharmacokinetic properties of Bdomain deleted recombinant FVIII (rBDD-FVIII), rFVIIIFc, FVIII-169:Fcand FVIII-169:VWF-031 were evaluated in FVIII/VWF double knockout (DKO)mice.

DKO mice were treated with a single intra venous administration of 200IU/kg of FVIII proteins, and plasma samples were collected at designatedtime points as indicated in FIG. 11. FVIII activity of the plasmasamples were analyzed by FVIII chromogenic assay and half-life wascalculated using the WinNonlin-Phoenix program. The pharmacokineticparameters of the tested molecules are listed in Table 21. The timeregression curve of plasma FVIII activity for each FVIII variants wereplotted in FIG. 11.

Unmodified BDD-FVIII had a half-life of 0.23 hr in DKO mice, the FVIIIFcfusion protein has an extended half-life of 1.66 hr in DKO mice due tothe recycling of FVIIIFc protein through the Fc:FcRn interaction. When a288 residue of AEXTEN polypeptide was incorporated into the B domainregion of FVIII within the FVIIIFc molecule, the half-life of theresulting FVIII169/Fc protein was further extended to 7.41 hr in DKOmice. Finally, with the addition of the D′D3 domain of VWF, thehalf-life of FVIII169NWF031 heterodimer has reached 17.9 hr in DKO mice(FIG. 11, Table 21). In addition of the half-life, all of the other PKparameters also improved proportionally with the addition of eachelement (Table 21). FVIII can tolerate multiple half-life extensionelements, and this synergistic effect of the three elements on FVIIIhalf-life extension, enabled the further improvement of the half-life ofFVIII-XTEN VWF heterodimers.

TABLE 21 PK parameters of FVIII variants XTEN Insertions Cl AUC_D FVIIIXTEN T_(1/2) MRT (mL/hr/ Vss kg*hr/ FVIII Isoform Site Length (hr) (hr)kg) (mL/kg) mL BDD-FVIII dc 0.23 0.24 407.72 97.42 0.0025 FVIIIFc dc1.66 2.06 62.66 128.82 0.0161 FVIII169/Fc se B* AE288 7.41 6.67 6.2441.61 0.1603 FVIII169/VWF031 sc B* AE288 17.94 20.1 4.06 81.69 0.2463 *Bindicates an XTEN sequence (e.g., 144) is inserted immediatelydownstream of amino acid residue 745 corresponding to mature FVIIIsequence.

Example 12: Pharmacokinetic Properties of Different FVIII-XTEN VWFHeterodimers

To evaluate the combined effect of the VWF-D′D3 fragment and XTENinsertions on the FVIII half-life, the pharmacokinetic properties ofFVIII-XTEN-Fc:VWF-Fc heterodimers were tested in HemA mice and comparedto those of the single chain isoform of BDD-FVIII (scBDD-FVIII) andFVIII-169:VWF-031 (example 10). Seven new FVIII-XTEN-Fc constructs weregenerated (protein sequences were listed in Table 24). Schematicdiagrams of those constructs are shown in FIG. 14A-H. FVIII-195 andFVIII-199, respectively, are the FVIII dual chain and single chainisoforms that each contains two XTEN insertions at positions 1900 and1656. FVIII-196 and FVIII-201, respectively, are the FVIII dual chainand single chain isoforms that each contains three XTEN insertions atpositions 26, 1656 and 1900. FVIII-203, -204 and -205 are sc-FVIIIFcmolecules with two XTEN insertions at the B domain junction and atpositions 1900, 403 or 18, respectively. Each FVIII-XTEN-Fc constructwas co-expressed with VWF-031 in HEK293 cells to produceFVIII-XTEN-Fc/VWF heterodimeric proteins. At day four post-transfection,cell culture medium was harvested and either concentrated to 20 IU/mLbased on FVIII chromogenic activity (FVIII-195:VWF-031,FVIII-196:VWF-031, FVIII-199:VWF-031, FVIII-203:VWF-031 andFVIII-204:VWF-031) or purified (scBDD-FVIII, FVIII-169:VWF-031,FVIII-201:VWF-031 and FVIII-205:VWF-031). Having demonstrated thecomplete intra-molecular shielding of FVIII molecule from the endogenousVWF by the D′D3 fragment in the FVIII-XTEN-Fc:VWF-Fc heterodimer(FVIII-169:VWF-031, Example 5), HemA mice was chosen for the PKevaluations. Purified protein or concentrated cell culture medium wasadministered to 8-12 week-old HemA mice by intravenous administration ata dose of 200 IU/10 mL/kg. Plasma samples were collected at 5 min, 8 hr,16 hr, 24 hr, 32 hr, 48 hr, 72 hr and 96 hr post-dosing. FVIII activityof the plasma samples were analyzed by FVIII chromogenic assay andhalf-life was calculated using the WinNonlin-Phoenix program. Thepharmacokinetic parameters of the tested molecules are listed in Table22. The plasma FVIII activities at selected time points forFVIII-XTEN-Fc/VWF-Fc variants were plotted in FIGS. 12A-C.

When XTEN was inserted into positions 1900 and 1656 (FVIII-195,FVIII-199), moderate improvement in half-life was observed for thescFVIII isoform (FVIII-199:VWF-031) compared to FVIII-169:VWF-031.However, the dcFVIII isoform exhibited a shorter half-life than didFVIII-169:VWF-031, indicating that the single chain isoform might besignificantly more stable than the corresponding dual chain isoform(Table 22 and FIG. 12A). When a third XTEN insertion was incorporatedinto FVIII-199 at position 26, the half-life of the resulting moleculeFVIII-201:VWF-031 had reached 24.6 hr, which represents greater than athreefold half-life improvement relative to scBDD-FVIII (Table 22 andFIG. 12C). We have also tested the half-life extension effect of thesecond XTEN insertion at position 403 (A2 domain), 1900 (A3 domain) and18 (A1 domain) each in combination with the B domain XTEN insertion.While the addition of the A2 or A3 XTEN insertion did not confer anadditional half-life benefit (Table 22, FIG. 12b ), the addition of theA1 insertion further extended the half-life of the FVIII-XTEN-Fc:VWF-Fcheterodimer to 29.4 hr (Table 22, FIG. 12C), which is greater thanthreefold longer than that of scBDD-FVIII.

When XTENs were incorporated into the FVIIIFc/VWF heterodimer construct,degree of half-life improvement of the resulting molecules was variable,and no obvious correlation was observed between half-lives and eitherthe site or number of XTEN insertion, suggesting that the half-life ofthe FVIII-XTEN-Fc/VWF heterodimer is determined by the integrity of thewhole molecule rather than by the number or placement of XTENinsertions.

The 24.6 hr and 29.4 hr half-lives observed for FVIII-XTEN-Fc:VWF-Fcheterodimers clearly exceeded the 1.6- to 2-fold limitation on FVIIIhalf-life extension. If this finding translates for HemA patients, itwill allow once-weekly or less frequent dosing for FVIII prophylaxis.

TABLE 22 PK parameters of FVIII-XTEN-Fc/VWF-Fc heterodimers Cl AUC_DFVIII XTEN Insertions T_(1/2) MRT (mL/hr/ Vss kg*hr/ FVIII Isoform SiteXTEN Length (hr) (hr) kg) (mL/kg) mL scBDD-FVIII sc 7.16 10.16 4.3844.44 0.23 FVIII169/VWF031 sc B* AE288 16.65 18.44 3.57 65.79 0.28FVIII195/VWF031 dc 1656/1900 AG144/ 12.56 13.88 9.04 125.48 0.11 AE144FV111199/VWF031 sc 1656/1900 AG144/ 18.57 20.09 6.24 125.28 0.16 AE144FVIII201/VWF031 sc 26/1656/ AG144/AG144/ 24.63 33.67 1.9 63.97 0.53 1900AE144 FVIII203/VWF031 sc  403/B* AE144/AE288 15.52 18 3.65 65.61 0.27FVIII204/VWF031 sc 1900/B* AE144/AE288 16.3 20.63 2.87 59.14 0.35FVIII205/VWF031 sc  18/B* AE144/AE288 29.4 37.06 1.82 67.39 0.55 *Bindicates an XTEN sequence (e.g., 144) is inserted immediatelydownstream of amino acid residue 745 corresponding to mature FVIIIsequence.

In addition to incorporating XTEN into the FVIII molecule, we alsoevaluated the potential half-life extension benefit of incorporatingXTEN as a linker between the D′D3 and Fc fragment. FVIII-155 (scFVIIIFc)was co-expressed with VWF-034 (VWF-Fc with AE 288 XTEN plus a 35 residuethrombin cleavable linker) in HEK293 cells. At day 4 post-transfection,cell culture medium was harvested and concentrated to 20 IU/mL based onFVIII activity assay. FVIII/VWF DKO mice were dosed with concentratedcell culture media at 200 IU/10 mL/kg with a single intravenousinjection. Plasma samples were collected at 5 min, 8 hr, 24 hr, 48 hr,72 hr and 96 hr post-dosing. The FVIII activity of plasma samples wasanalyzed by FVIII chromogenic assay, and the regression curve of plasmaFVIII activity as a function of time was plotted (FIG. 13).FVIII-155/VWF-034 exhibited the same improvement in half-life asFVIII-169/VWF-031, which has AE 288 XTEN inserted into the B domainjunction of FVIII, as illustrated by the over lapping regression curvesfor the two molecules (FIG. 13). The demonstration that XTEN insertioninto the VWF-Fc polypeptide confers half-life improvement of a magnitudesimilar to that conferred by XTEN insertion at the B domain junction ofthe FVIII polypeptide suggests that further half-life improvement may bepossible in a heterodimeric molecule in which intra-molecular XTENinsertion in the FVIII polypeptide is combined with inter-domain XTENinsertion between the VWF and Fc elements of the VWF-Fc polypeptide.

Example 13A: Pharmacokinetic Properties of Additional FVIII-XTEN VWFHeterodimers

In addition to the FVIII-XTEN VWF heterodimers that were listed in Table22, FVIII-XTEN VWF heterodimers containing different composition of XTENinsertions, single chain and dual chain version of FVIII (Table 23A) areeither tested or will be tested in HemA for their pharmacokineticproperties. Various FVIII constructs (Table 23B) and VWF constructs(Table 23C) are also disclosed below. HemA mice will be treated with asingle dose of intravenous administration of the heterodimer proteins at200 IU/10 mL/kg. Plasma samples will then be collected at 5 min, 24, 48,72, 96 and 120 hrs post-dosing. FVIII activity of the plasma sampleswill be analyzed by FVIII chromogenic assay and half-life will becalculated using the WinNonlin-Phoenix program. The protein sequences ofthe listed heterodimers were listed in Table 25.

TABLE 23A Plausible FVIII-XTEN-Fc:VVVF-Fc heterodimer combinations forPK and activity improvement. pSYN pSYN VWF- pSYN VWF- pSYN VWF-015 031034 ** VWF-036 pSYN FVIII — t_(1/2) 8.7 hr To be tested — 010 DKO micepSYN FVIII t_(1/2) 6.3 hr t_(1/2) 10.8 hr t_(1/2) 18.6 hr t_(1/2) 13.3hr 155 DKO mice HemA mice HemA mice HemA mice pSYN FVIII — t_(1/2) 16.7hr t_(1/2) 31.1 hr — 169 ** HemA mice HemA mice pSYN FVIII — t_(1/2)15.2 hr t_(1/2) 28.9 hr To be tested 173 ** DKO mice HemA mice pSYNFVIII — t_(1/2) 29.4 hr t_(1/2) 32.4 hr t_(1/2) 29.7 hr 205 HemA miceHemA mice HemA mice pSYN FVIII — t_(1/2) 24.5 hr t_(1/2) 27.4 hr — 266HemA mice HemA mice pSYN FVII — t_(1/2) 23.0 hr t_(1/2) 25.7 hr 267 HemAmice HemA mice pSYN FVIII — To be tested To be tested To be tested 268Dual chain To be tested To be tested To be tested isoform of pSYN FVIII268 ** Length of XTEN can be changed to 72, 144, 288, 324, 333, 576, or864.

TABLE 23B FVIII Constructs: pSYN FVIII dual chain FVIIIFc 010 pSYN FVIIISingle chain FVIIIFc with 288 AE XTEN in B- 169 domain pSYN FVIII dualchain FVIIIFc with 288 AE XTEN in B- 173 domain pSYN FVIII dual chainFVIIIFc with two 144 XTENs at amino 195 acid 1656 and 1900 pSYN FVIIIdual chain FVIIIFc with three 144 XTENs at amino 196 acid 26, 1656 and1900 pSYN FVIII Single chain FVIIIFc with two 144 XTENs at amino 199acid 1656 and 1900 pSYN FVIII Single chain FVIIIFc with three 144 XTENsat 201 amino acid 26, 1656 and 1900 pSYN FVIII Single chain FVIIIFc with144 AE XTEN at amino 203 acid 1900 and 288 AE XTEN in B-domain pSYNFVIII Single chain FVIIIFc with 144 AE XTEN at amino 204 acid 403 and288 AE XTEN in B-domain pSYN FVIII Single chain FVIIIFc with 144 AE XTENat amino 205 acid 18 and 288 AE XTEN in B-domain pSYN FVIII Single chainFVIII (no Fc, no XTEN) 207 pSYN FVIII Single chain FVIIIFc with 42 AEXTEN at amino 266 acid 18 and 288 AE XTEN in B-domain pSYN FVIII Singlechain FVIIIFc with 72 AE XTEN at amino 267 acid 18 and 288 AE XTEN inB-domain pSYN FVIII Single chain FVIIIFc with 144 AE XTEN at amino 268acid 18 pSYN FVIII Single chain FVIIIFc with 72 AE XTEN at amino 269acid 18 pSYN FVIII Single chain FVIIIFc with 42 AE XTEN at amino 271acid 18 pSYN FVIII Single chain FVIII with 144 AE XTEN at amino 272 acid18 and 288 AE XTEN in B-domain (no Fc)

TABLE 23C VWF Constructs: pSYN VWF-D1D2D′D3- 48aa long thrombincleavable GS VWF031 linker-Fc with C1099A/C1142A pSYN VWF-D1D2D′D3-288AE XTEN +35aa long thrombin VWF034 cleavable GS linker-Fc withC1099A/C1142A pSYN VWF-D1D2D′D3- 72aa long thrombin cleavable GSl VWF035inker-Fc with C1099A/C1142A pSYN VWF-D1D2D′D3- 98aa long thrombincleavable GS VWF036 linker-Fc with C1099A/C1142A pSYN VWF-D1D2D′D3 with288 AE XTEN in D3 and 48aa VWF041 long thrombin cleavable GS linkerafter D3-Fc with C1099A/C1142A

Example 13B: Pharmacokinetic Properties of Additional FVIII-XTEN VWFHeterodimers

FVIII-XTEN VWF heterodimers were tested in HemA mice for theirpharmacokinetic properties. The heterodimers tested are FVIII169NWF034,FVIII205NWF034, FVIII205NWF036 and FVII1266NWF031. HemA mice wereadministered with a single intravenous dose of various heterodimerproteins at 200 IU/10 mL/kg. Plasma samples were collected at 5 min, 24,48, 72, 96 and 120 hrs post-dosing. FVIII activity of the plasma sampleswere analyzed by FVIII chromogenic assay, and half-lives were calculatedusing the WinNonlin-Phoenix program. The PK results are shown below inTable 24.

TABLE 24 Additional FVIII-XTEN_VWF - PK in HemA Mice Fold of t_(1/2)AUC_D increase 5 min Cl Vss (hr*kg* vs recovery HL MRT (mL/hr/ (mL/ mIU/scBDD- Treatment (%) (hr) (hr) kg) kg) mL/mIU) FVIII ScBDD- 7.16 10.164.83 44.44 0.23 — FVIII FVIII169/ 76 31.1 34.57 1.73 59.77 0.58 4.3VWF034 FVIII205/ 68 32.41 39.79 1.55 61.73 0.64 4.6 VWF034 FVIII205/ 7429.71 36.35 1.61 58.43 0.62 4.1 VWF036 FVIII266/ 66 24.45 22.75 2.6760.83 0.37 3.4 VWF031

pSYNFVIII 010 nucleotide sequence-(Dual chain FVIIIFc) (SEQ ID NO: 125)   1 ATGCAAATAG AGCTCTCCAC CTGCTTCTTT CTGTGCCTTT TGCGATTCTG   51CTTTAGTGCC ACCAGAAGAT ACTACCTGGG TGCAGTGGAA CTGTCATGGG  101ACTATATGCA AAGTGATCTC GGTGAGCTGC CTGTGGACGC AAGATTTCCT  151CCTAGAGTGC CAAAATCTTT TCCATTCAAC ACCTCAGTCG TGTACAAAAA  201GACTCTGTTT GTAGAATTCA CGGATCACCT TTTCAACATC GCTAAGCCAA  251GGCCACCCTG GATGGGTCTG CTAGGTCCTA CCATCCAGGC TGAGGTTTAT  301GATACAGTGG TCATTACACT TAAGAACATG GCTTCCCATC CTGTCAGTCT  351TCATGCTGTT GGTGTATCCT ACTGGAAAGC TTCTGAGGGA GCTGAATATG  401ATGATCAGAC CAGTCAAAGG GAGAAAGAAG ATGATAAAGT CTTCCCTGGT  451GGAAGCCATA CATATGTCTG GCAGGTCCTG AAAGAGAATG GTCCAATGGC  501CTCTGACCCA CTGTGCCTTA CCTACTCATA TCTTTCTCAT GTGGACCTGG  551TAAAAGACTT GAATTCAGGC CTCATTGGAG CCCTACTAGT ATGTAGAGAA  601GGGAGTCTGG CCAAGGAAAA GACACAGACC TTGCACAAAT TTATACTACT  651TTTTGCTGTA TTTGATGAAG GGAAAAGTTG GCACTCAGAA ACAAAGAACT  701CCTTGATGCA GGATAGGGAT GCTGCATCTG CTCGGGCCTG GCCTAAAATG  751CACACAGTCA ATGGTTATGT AAACAGGTCT CTGCCAGGTC TGATTGGATG  801CCACAGGAAA TCAGTCTATT GGCATGTGAT TGGAATGGGC ACCACTCCTG  851AAGTGCACTC AATATTCCTC GAAGGTCACA CATTTCTTGT GAGGAACCAT  901CGCCAGGCGT CCTTGGAAAT CTCGCCAATA ACTTTCCTTA CTGCTCAAAC  951ACTCTTGATG GACCTTGGAC AGTTTCTACT GTTTTGTCAT ATCTCTTCCC 1001ACCAACATGA TGGCATGGAA GCTTATGTCA AAGTAGACAG CTGTCCAGAG 1051GAACCCCAAC TACGAATGAA AAATAATGAA GAAGCGGAAG ACTATGATGA 1101TGATCTTACT GATTCTGAAA TGGATGTGGT CAGGTTTGAT GATGACAACT 1151CTCCTTCCTT TATCCAAATT CGCTCAGTTG CCAAGAAGCA TCCTAAAACT 1201TGGGTACATT ACATTGCTGC TGAAGAGGAG GACTGGGACT ATGCTCCCTT 1251AGTCCTCGCC CCCGATGACA GAAGTTATAA AAGTCAATAT TTGAACAATG 1301GCCCTCAGCG GATTGGTAGG AAGTACAAAA AAGTCCGATT TATGGCATAC 1351ACAGATGAAA CCTTTAAGAC TCGTGAAGCT ATTCAGCATG AATCAGGAAT 1401CTTGGGACCT TTACTTTATG GGGAAGTTGG AGACACACTG TTGATTATAT 1451TTAAGAATCA AGCAAGCAGA CCATATAACA TCTACCCTCA CGGAATCACT 1501GATGTCCGTC CTTTGTATTC AAGGAGATTA CCAAAAGGTG TAAAACATTT 1551GAAGGATTTT CCAATTCTGC CAGGAGAAAT ATTCAAATAT AAATGGACAG 1601TGACTGTAGA AGATGGGCCA ACTAAATCAG ATCCTCGGTG CCTGACCCGC 1651TATTACTCTA GTTTCGTTAA TATGGAGAGA GATCTAGCTT CAGGACTCAT 1701TGGCCCTCTC CTCATCTGCT ACAAAGAATC TGTAGATCAA AGAGGAAACC 1751AGATAATGTC AGACAAGAGG AATGTCATCC TGTTTTCTGT ATTTGATGAG 1801AACCGAAGCT GGTACCTCAC AGAGAATATA CAACGCTTTC TCCCCAATCC 1851AGCTGGAGTG CAGCTTGAGG ATCCAGAGTT CCAAGCCTCC AACATCATGC 1901ACAGCATCAA TGGCTATGTT TTTGATAGTT TGCAGTTGTC AGTTTGTTTG 1951CATGAGGTGG CATACTGGTA CATTCTAAGC ATTGGAGCAC AGACTGACTT 2001CCTTTCTGTC TTCTTCTCTG GATATACCTT CAAACACAAA ATGGTCTATG 2051AAGACACACT CACCCTATTC CCATTCTCAG GAGAAACTGT CTTCATGTCG 2101ATGGAAAACC CAGGTCTATG GATTCTGGGG TGCCACAACT CAGACTTTCG 2151GAACAGAGGC ATGACCGCCT TACTGAAGGT TTCTAGTTGT GACAAGAACA 2201CTGGTGATTA TTACGAGGAC AGTTATGAAG ATATTTCAGC ATACTTGCTG 2251AGTAAAAACA ATGCCATTGA ACCAAGAAGC TTCTCTCAAA ACCCACCAGT 2301CTTGAAACGC CATCAACGGG AAATAACTCG TACTACTCTT CAGTCAGATC 2351AAGAGGAAAT TGACTATGAT GATACCATAT CAGTTGAAAT GAAGAAGGAA 2401GATTTTGACA TTTATGATGA GGATGAAAAT CAGAGCCCCC GCAGCTTTCA 2451AAAGAAAACA CGACACTATT TTATTGCTGC AGTGGAGAGG CTCTGGGATT 2501ATGGGATGAG TAGCTCCCCA CATGTTCTAA GAAACAGGGC TCAGAGTGGC 2551AGTGTCCCTC AGTTCAAGAA AGTTGTTTTC CAGGAATTTA CTGATGGCTC 2601CTTTACTCAG CCCTTATACC GTGGAGAACT AAATGAACAT TTGGGACTCC 2651TGGGGCCATA TATAAGAGCA GAAGTTGAAG ATAATATCAT GGTAACTTTC 2701AGAAATCAGG CCTCTCGTCC CTATTCCTTC TATTCTAGCC TTATTTCTTA 2751TGAGGAAGAT CAGAGGCAAG GAGCAGAACC TAGAAAAAAC TTTGTCAAGC 2801CTAATGAAAC CAAAACTTAC TTTTGGAAAG TGCAACATCA TATGGCACCC 2851ACTAAAGATG AGTTTGACTG CAAAGCCTGG GCTTATTTCT CTGATGTTGA 2901CCTGGAAAAA GATGTGCACT CAGGCCTGAT TGGACCCCTT CTGGTCTGCC 2951ACACTAACAC ACTGAACCCT GCTCATGGGA GACAAGTGAC AGTACAGGAA 3001TTTGCTCTGT TTTTCACCAT CTTTGATGAG ACCAAAAGCT GGTACTTCAC 3051TGAAAATATG GAAAGAAACT GCAGGGCTCC CTGCAATATC CAGATGGAAG 3101ATCCCACTTT TAAAGAGAAT TATCGCTTCC ATGCAATCAA TGGCTACATA 3151ATGGATACAC TACCTGGCTT AGTAATGGCT CAGGATCAAA GGATTCGATG 3201GTATCTGCTC AGCATGGGCA GCAATGAAAA CATCCATTCT ATTCATTTCA 3251GTGGACATGT GTTCACTGTA CGAAAAAAAG AGGAGTATAA AATGGCACTG 3301TACAATCTCT ATCCAGGTGT TTTTGAGACA GTGGAAATGT TACCATCCAA 3351AGCTGGAATT TGGCGGGTGG AATGCCTTAT TGGCGAGCAT CTACATGCTG 3401GGATGAGCAC ACTTTTTCTG GTGTACAGCA ATAAGTGTCA GACTCCCCTG 3451GGAATGGCTT CTGGACACAT TAGAGATTTT CAGATTACAG CTTCAGGACA 3501ATATGGACAG TGGGCCCCAA AGCTGGCCAG ACTTCATTAT TCCGGATCAA 3551TCAATGCCTG GAGCACCAAG GAGCCCTTTT CTTGGATCAA GGTGGATCTG 3601TTGGCACCAA TGATTATTCA CGGCATCAAG ACCCAGGGTG CCCGTCAGAA 3651GTTCTCCAGC CTCTACATCT CTCAGTTTAT CATCATGTAT AGTCTTGATG 3701GGAAGAAGTG GCAGACTTAT CGAGGAAATT CCACTGGAAC CTTAATGGTC 3751TTCTTTGGCA ATGTGGATTC ATCTGGGATA AAACACAATA TTTTTAACCC 3801TCCAATTATT GCTCGATACA TCCGTTTGCA CCCAACTCAT TATAGCATTC 3851GCAGCACTCT TCGCATGGAG TTGATGGGCT GTGATTTAAA TAGTTGCAGC 3901ATGCCATTGG GAATGGAGAG TAAAGCAATA TCAGATGCAC AGATTACTGC 3951TTCATCCTAC TTTACCAATA TGTTTGCCAC CTGGTCTCCT TCAAAAGCTC 4001GACTTCACCT CCAAGGGAGG AGTAATGCCT GGAGACCTCA GGTGAATAAT 4051CCAAAAGAGT GGCTGCAAGT GGACTTCCAG AAGACAATGA AAGTCACAGG 4101AGTAACTACT CAGGGAGTAA AATCTCTGCT TACCAGCATG TATGTGAAGG 4151AGTTCCTCAT CTCCAGCAGT CAAGATGGCC ATCAGTGGAC TCTCTTTTTT 4201CAGAATGGCA AAGTAAAGGT TTTTCAGGGA AATCAAGACT CCTTCACACC 4251TGTGGTGAAC TCTCTAGACC CACCGTTACT GACTCGCTAC CTTCGAATTC 4301ACCCCCAGAG TTGGGTGCAC CAGATTGCCC TGAGGATGGA GGTTCTGGGC 4351TGCGAGGCAC AGGACCTCTA CGACAAAACT CACACATGCC CACCGTGCCC 4401AGCTCCAGAA CTCCTGGGCG GACCGTCAGT CTTCCTCTTC CCCCCAAAAC 4451CCAAGGACAC CCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTG 4501GTGGACGTGA GCCACGAAGA CCCTGAGGTC AAGTTCAACT GGTACGTGGA 4551CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG GAGCAGTACA 4601ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG 4651CTGAATGGCA AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC 4701CCCCATCGAG AAAACCATCT CCAAAGCCAA AGGGCAGCCC CGAGAACCAC 4751AGGTGTACAC CCTGCCCCCA TCCCGGGATG AGCTGACCAA GAACCAGGTC 4801AGCCTGACCT GCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGA 4851GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC ACGCCTCCCG 4901TGTTGGACTC CGACGGCTCC TTCTTCCTCT ACAGCAAGCT CACCGTGGAC 4951AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA 5001GGCTCTGCAC AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA 5051 AATGApSYNFVIII 010 protein sequence-(Dual chain FVIIIFc) (SEQ ID NO: 126)   1 MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP   51PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY  101DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG  151GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE  201GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM  251HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH  301RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE  351EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT  401WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY  451TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT  501DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR  551YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE  601NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL  651HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS  701MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL  751SKNNAIEPRS FSQNPPVLKR HQREITRTTL QSDQEEIDYD DTISVEMKKE  801DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG  851SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF  901RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP  951TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1001FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI 1051MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1101YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1151GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1201LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1251FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1301MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1351PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1401QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1451CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1501VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 1551LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 1601SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 1651KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

Example 14: A New Class of Coagulation Factor VIII Molecules withGreater than Three-Fold Half-Life Extension in Hemophilia a Mice

The new class of FVIII molecules was designed to contain twopolypeptides; one that consists of a single chain B-domain deleted (BDD)FVIII with XTEN inserted at one or more locations within the FVIIIsequence, and one that is composed of the D′D3 region of VWF. Eachpolypeptide was also recombinantly fused to the Fc region of IgG1 toenable the D′D3 region to be correctly aligned to bind the FVIII moiety.The resulting FVIII variants were expressed in HEK 293 cells bytransient transfection, and purified from the conditioned media. FVIIIactivity was evaluated by FVIII chromogenic assay and thepharmacokinetic properties were assessed in both FVIII knockout (HemA)and FVIII/VWF double knock-out (DKO) mice.

Incorporating XTEN and D′D3 region of VWF into rFVIII led to theuncoupling of the clearance of the fusion proteins from endogenous VWFwhile extending their circulating half-life. FVIII in this fusionconfiguration is completely shielded from interacting with VWF, asmeasured by biolayer interferometry (Octet) analysis. Consistent withthis, their pharmacokinetic profiles in HemA and DKO mice were found tobe identical, indicating that their clearance rate in mice waseffectively disconnected from VWF. Optimization of XTEN length and thelocations for inserting XTEN identified a subset of the proteins thathave exceeded the VWF limitation (16 hours), reaching a circulatinghalf-life of up to 30 hours in HemA mice representing a 4-foldimprovement over BDD-FVIII. Importantly, these proteins maintained theirfunctionality, as judged by FVIII chromogenic assay.

The VWF dependency has set a fundamental limitation for half-life oftherapeutic FVIII. Uncoupling FVIII from VWF clearance pathways whileextending half-life by the fusion of D′D3 region of VWF and XTEN hasgenerated a novel FVIII molecule with a 4-fold half-life extension. Thisis the first report of an engineered FVIII that has exceeded thehalf-life limitation observed through industry-wide efforts indevelopment of long-lasting FVIII, representing a potentiallysignificant advancement in prophylactic treatment of hemophilia A.

TABLE 25 Protein sequences of FVIII-XTEN-Fc and VWF-Fc constructs FVIII195 protein sequence (dual chain FVIIIFc with two 144 AE XTENs atamino acid 1656 and 1900) (SEQ ID NO: 105)    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP   51PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY  101DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG  151GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE  201GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM  251HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH  301RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE  351EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT  401WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY  451TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT  501DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLIR  551YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE  601NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL  651HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS  701MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL  751SKNNAIEPRS FSQNPPVLKR HQREITRTTL QGAPGTPGSG TASSSPGASP  801GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST GTGPGTPGSG  851TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSTPSGA  901TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD DTISVEMKKE  951DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 1001SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 1051RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 1101TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1151FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG SEPATSGSET 1201PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG 1251TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG SPTSTEEGTS 1301ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN YRFHAINGYI 1351MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1401YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1451GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1501LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1551FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1601MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1651PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1701QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1751CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1801VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 1851LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 1901SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 1951KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*FVIII 196 protein sequence (dual chain FVIIIFc with three 144 AEXTENs at amino add 26, 1656 and 1900) (SEQ ID NO: 106)    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVGAPGS   51SPSASTGTGP GSSPSASTGT GPGASPGTSS TGSPGASPGT SSTGSPGSST  101PSGATGSPGS SPSASTGTGP GASPGTSSTG SPGSSPSAST GTGPGTPGSG  151TASSSPGSST PSGATGSPGS STPSGATGSP GASPGTSSTG SPASSDARFP  201PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY  251DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG  301GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE  351GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM  401HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH  451RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE  501EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT  551WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY  601TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT  651DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR  701YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE  751NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL  801HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS  851MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL  901SKNNAIEPRS FSQNPPVLKR HQREITRTTL QGAPGTPGSG TASSSPGASP  951GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST GTGPGTPGSG 1001TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSTPSGA 1051TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD DTISVEMKKE 1101DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 1151SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 1201RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 1251TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1301FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG SEPATSGSET 1351PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG 1401TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG SPTSTEEGTS 1451ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN YRFHAINGYI 1501MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1551YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1601GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1651LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1701FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1751MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1801PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1851QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1901CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1951VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 2001LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 2051SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 2101KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*FVIII 199 protein sequence (single chain FVIIIFc with three 144AE XTENs at amino acid 1656 and 1900) (SEO ID NO: 107)    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP   51PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY  101DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG  151GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE  201GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM  251HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH  301RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE  351EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT  401WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY  451TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT  501DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR  551YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE  601NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL  651HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS  701MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL  751SKNNAIEPRS FSQNPPVLKR HQAEITRTTL QGAPGTPGSG TASSSPGASP  801GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST GTGPGTPGSG  851TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSTPSGA  901TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD DTISVEMKKE  951DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 1001SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 1051RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 1101TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1151FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG SEPATSGSET 1201PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG 1251TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG SPTSTEEGTS 1301ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN YRFHAINGYI 1351MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1401YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1451GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1501LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1551FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1601MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1651PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1701QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1751CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1801VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 1851LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 1901SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 1951KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*FVIII 201 protein sequence (single chain FVIIIFc with three 144AE XTENs at amino acid 26, 1656 & 1900) (SEQ ID NO: 108)    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVGAPGS   51SPSASTGTGP GSSPSASTGT GPGASPGTSS TGSPGASPGT SSTGSPGSST  101PSGATGSPGS SPSASTGTGP GASPGTSSTG SPGSSPSAST GTGPGTPGSG  151TASSSPGSST PSGATGSPGS STPSGATGSP GASPGTSSTG SPASSDARFP  201PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY  251DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG  301GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE  351GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM  401HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH  451RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE  501EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT  551WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY  601TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT  651DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR  701YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE  751NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL  801HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS  851MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL  901SKNNAIEPRS FSQNPPVLKR HQAEITRTTL QGAPGTPGSG TASSSPGASP  951GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSPSAST GTGPGTPGSG 1001TASSSPGASP GTSSTGSPGA SPGTSSTGSP GASPGTSSTG SPGSSTPSGA 1051TGSPGSSTPS GATGSPGASP GTSSTGSPAS SSDQEEIDYD DTISVEMKKE 1101DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 1151SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 1201RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 1251TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1301FALFFTIFDE TKSWYFTENM ERNCRGAPTS ESATPESGPG SEPATSGSET 1351PGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG 1401TSESATPESG PGSPAGSPTS TEEGSPAGSP TSTEEGSPAG SPTSTEEGTS 1451ESATPESGPG TSTEPSEGSA PGASSAPCNI QMEDPTFKEN YRFHAINGYI 1501MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1551YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1601GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1651LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1701FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1751MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1801PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1851QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1901CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1951VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 2001LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 2051SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 2101KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*FVIII 203 protein sequence (single chain FVIIIFc with two AE XTENs;one 288AE XTEN in B-domain and one 144 AE XTEN at amino acid 1900)(SEQ ID NO: 109)    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP   51PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY  101DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG  151GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE  201GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM  251HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH  301RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE  351EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT  401WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY  451TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT  501DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR  551YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE  601NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL  651HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS  701MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL  751SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE  801SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE  851EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG  901SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS  951ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE 1001PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS 1051EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD 1101EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK 1151KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR 1201PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD 1251CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT 1301IFDETKSWYF TENMERNCRG APTSESATPE SGPGSEPATS GSETPGTSES 1351ATPESGPGSE PATSGSETPG TSESATPESG PGTSTEPSEG SAPGTSESAT 1401PESGPGSPAG SPTSTEEGSP AGSPTSTEEG SPAGSPTSTE EGTSESATPE 1451SGPGTSTEPS EGSAPGASSA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG 1501LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG 1551VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH 1601IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII 1651HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD 1701SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME 1751SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ 1801VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK 1851VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL 1901YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE 1951DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 2001KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV 2051KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 2101GNVFSCSVMH EALHNHYTQK SLSLSPGK*FVIII 204 protein sequence (single chain FVIIIFc with two AE XTENs;one 288AE XTEN in B-domain and one 144 AE XTEN at amino acid 403)(SEQ ID NO: 110)    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP   51PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY  101DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG  151GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE  201GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM  251HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH  301RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE  351EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT  401WVHYIAAEEE DWDYAPLVLA PDGAPTSTEP SEGSAPGSPA GSPTSTEEGT  451STEPSEGSAP GTSTEPSEGS APGTSESATP ESGPGTSTEP SEGSAPGTSE  501SATPESGPGS EPATSGSETP GTSTEPSEGS APGTSTEPSE GSAPGTSESA  551TPESGPGTSE SATPESGPGA SSDRSYKSQY LNNGPQRIGR KYKKVRFMAY  601TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT  651DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR  701YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE  751NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL  801HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS  851MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL  901SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE  951SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE 1001EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG 1051SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS 1101ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE 1151PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS 1201EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD 1251EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK 1301KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR 1351PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD 1401CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT 1451IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG 1501LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG 1551VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH 1601IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII 1651HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD 1701SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME 1751SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ 1801VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK 1851VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL 1901YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE 1951DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 2001KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV 2051KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 2101GNVFSCSVMH EALHNHYTQK SLSLSPGK*FVIII 205 protein sequence (single chain FVIIIFc with two AE XTENs;one 288AE XTEN in B-domain and one 144 AE XTEN at amino acid 18)(SEQ ID NO: 111)    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG   51PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG  101TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS  151ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL GELPVDARFP  201PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY  251DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG  301GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE  351GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM  401HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH  451RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE  501EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT  551WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY  601TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT  651DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR  701YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE  751NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL  801HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS  851MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL  901SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE  951SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE 1001EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG 1051SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS 1101ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE 1151PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS 1201EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD 1251EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK 1301KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR 1351PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD 1401CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT 1451IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG 1501LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG 1551VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH 1601IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII 1651HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD 1701SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME 1751SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ 1801VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK 1851VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL 1901YDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE 1951DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 2001KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV 2051KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 2101GNVFSCSVMH EALHNHYTQK SLSLSPGK*pSYN FVIII 266 protein sequence (FVIII Fc with 42 AE-XTEN at aminoacid 18 and 288 AE XTEN in B-domain) SEQ ID NO: 112    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP GSPAGSPTST   51EEGTSESATP ESGPGSEPAT SGSETPASSS DLGELPVDAR FPPRVPKSFP  101FNTSVVYKKT LFVEFTDHLF NIAKPRPPWM GLLGPTIQAE VYDTVVITLK  151NMASHPVSLH AVGVSYWKAS EGAEYDDQTS QREKEDDKVF PGGSHTYVWQ  201VLKENGPMAS DPLCLTYSYL SHVDLVKDLN SGLIGALLVC REGSLAKEKT  251QTLHKFILLF AVFDEGKSWH SETKNSLMQD RDAASARAWP KMHTVNGYVN  301RSLPGLIGCH RKSVYWHVIG MGTTPEVHSI FLEGHTFLVR NHRQASLEIS  351PITFLTAQTL LMDLGQFLLF CHISSHQHDG MEAYVKVDSC PEEPQLRMKN  401NEEAEDYDDD LTDSEMDVVR FDDDNSPSFI QIRSVAKKHP KTWVHYIAAE  451EEDWDYAPLV LAPDDRSYKS QYLNNGPQRI GRKYKKVRFM AYTDETFKTR  501EAIQHESGIL GPLLYGEVGD TLLIIFKNQA SRPYNIYPHG ITDVRPLYSR  551RLPKGVKHLK DFPILPGEIF KYKWTVTVED GPTKSDPRCL TRYYSSFVNM  601ERDLASGLIG PLLICYKESV DQRGNQIMSD KRNVILFSVF DENRSWYLTE  651NIQRFLPNPA GVQLEDPEFQ ASNIMHSING YVFDSLQLSV CLHEVAYWYI  701LSIGAQTDFL SVFFSGYTFK HKMVYEDTLT LFPFSGETVF MSMENPGLWI  751LGCHNSDFRN RGMTALLKVS SCDKNTGDYY EDSYEDISAY LLSKNNAIEP  801RSFSQNGAPG TSESATPESG PGSEPATSGS ETPGTSESAT PESGPGSEPA  851TSGSETPGTS ESATPESGPG TSTEPSEGSA PGSPAGSPTS TEEGTSESAT  901PESGPGSEPA TSGSETPGTS ESATPESGPG SPAGSPTSTE EGSPAGSPTS  951TEEGTSTEPS EGSAPGTSES ATPESGPGTS ESATPESGPG TSESATPESG 1001PGSEPATSGS ETPGSEPATS GSETPGSPAG SPTSTEEGTS TEPSEGSAPG 1051TSTEPSEGSA PGSEPATSGS ETPGTSESAT PESGPGTSTE PSEGSAPASS 1101PPVLKRHQAE ITRTTLQSDQ EEIDYDDTIS VEMKKEDFDI YDEDENQSPR 1151SFQKKTRHYF IAAVERLWDY GMSSSPHVLR NRAQSGSVPQ FKKVVFQEFT 1201DGSFTQPLYR GELNEHLGLL GPYIRAEVED NIMVTFRNQA SRPYSFYSSL 1251ISYEEDQRQG AEPRKNFVKP NETKTYFWKV QHHMAPTKDE FDCKAWAYFS 1301DVDLEKDVHS GLIGPLLVCH TNTLNPAHGR QVTVQEFALF FTIFDETKSW 1351YFTENMERNC RAPCNIQMED PTFKENYRFH AINGYIMDTL PGLVMAQDQR 1401IRWYLLSMGS NENIHSIHFS GHVFTVRKKE EYKMALYNLY PGVFETVEML 1451PSKAGIWRVE CLIGEHLHAG MSTLFLVYSN KCQTPLGMAS GHIRDFQITA 1501SGQYGQWAPK LARLHYSGSI NAWSTKEPFS WIKVDLLAPM IIHGIKTQGA 1551RQKFSSLYIS QFIIMYSLDG KKWQTYRGNS TGTLMVFFGN VDSSGIKHNI 1601FNPPIIARYI RLHPTHYSIR STLRMELMGC DLNSCSMPLG MESKAISDAQ 1651ITASSYFTNM FATWSPSKAR LHLQGRSNAW RPQVNNPKEW LQVDFQKTMK 1701VTGVTTQGVK SLLTSMYVKE FLISSSQDGH QWTLFFQNGK VKVFQGNQDS 1751FTPVVNSLDP PLLTRYLRIH PQSWVHQIAL RMEVLGCEAQ DLYDKTHTCP 1801PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW 1851YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA 1901LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI 1951AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV 2001MHEALHNHYT QKSLSLSPGK *pSYN FVIII 267 protein sequence (FVIII Fc with 72 AE-XTEN at aminoacid 18 and 288 AE XTEN in B-domain) SEQ ID NO: 113    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG   51PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG  101TSTEPSEGSA PGASSSDLGE LPVDARFPPR VPKSFPFNTS VVYKKTLFVE  151FTDHLFNIAK PRPPWMGLLG PTIQAEVYDT VVITLKNMAS HPVSLHAVGV  201SYWKASEGAE YDDQTSQREK EDDKVFPGGS HTYVWQVLKE NGPMASDPLC  251LTYSYLSHVD LVKDLNSGLI GALLVCREGS LAKEKTQTLH KFILLFAVFD  301EGKSWHSETK NSLMQDRDAA SARAWPKMHT VNGYVNRSLP GLIGCHRKSV  351YWHVIGMGTT PEVHSIFLEG HTFLVRNHRQ ASLEISPITF LTAQTLLMDL  401GQFLLFCHIS SHQHDGMEAY VKVDSCPEEP QLRMKNNEEA EDYDDDLTDS  451EMDVVRFDDD NSPSFIQIRS VAKKHPKTWV HYIAAEEEDW DYAPLVLAPD  501DRSYKSQYLN NGPQRIGRKY KKVRFMAYTD ETFKTREAIQ HESGILGPLL  551YGEVGDTLLI IFKNQASRPY NIYPHGITDV RPLYSRRLPK GVKHLKDFPI  601LPGEIFKYKW TVTVEDGPTK SDPRCLTRYY SSFVNMERDL ASGLIGPLLI  651CYKESVDQRG NQIMSDKRNV ILFSVFDENR SWYLTENIQR FLPNPAGVQL  701EDPEFQASNI MHSINGYVFD SLQLSVCLHE VAYWYILSIG AQTDFLSVFF  751SGYTFKHKMV YEDTLTLFPF SGETVFMSME NPGLWILGCH NSDFRNRGMT  801ALLKVSSCDK NTGDYYEDSY EDISAYLLSK NNAIEPRSFS QNGAPGTSES  851ATPESGPGSE PATSGSETPG TSESATPESG PGSEPATSGS ETPGTSESAT  901PESGPGTSTE PSEGSAPGSP AGSPTSTEEG TSESATPESG PGSEPATSGS  951ETPGTSESAT PESGPGSPAG SPTSTEEGSP AGSPTSTEEG TSTEPSEGSA 1001PGTSESATPE SGPGTSESAT PESGPGTSES ATPESGPGSE PATSGSETPG 1051SEPATSGSET PGSPAGSPTS TEEGTSTEPS EGSAPGTSTE PSEGSAPGSE 1101PATSGSETPG TSESATPESG PGTSTEPSEG SAPASSPPVL KRHQAEITRT 1151TLQSDQEEID YDDTISVEMK KEDFDIYDED ENQSPRSFQK KTRHYFIAAV 1201ERLWDYGMSS SPHVLRNRAQ SGSVPQFKKV VFQEFTDGSF TQPLYRGELN 1251EHLGLLGPYI RAEVEDNIMV TFRNQASRPY SFYSSLISYE EDQRQGAEPR 1301KNFVKPNETK TYFWKVQHHM APTKDEFDCK AWAYFSDVDL EKDVHSGLIG 1351PLLVCHTNTL NPAHGRQVTV QEFALFFTIF DETKSWYFTE NMERNCRAPC 1401NIQMEDPTFK ENYRFHAING YIMDTLPGLV MAQDQRIRWY LLSMGSNENI 1451HSIHFSGHVF TVRKKEEYKM ALYNLYPGVF ETVEMLPSKA GIWRVECLIG 1501EHLHAGMSTL FLVYSNKCQT PLGMASGHIR DFQITASGQY GQWAPKLARL 1551HYSGSINAWS TKEPFSWIKV DLLAPMIIHG IKTQGARQKF SSLYISQFII 1601MYSLDGKKWQ TYRGNSTGTL MVFFGNVDSS GIKHNIFNPP IIARYIRLHP 1651THYSIRSTLR MELMGCDLNS CSMPLGMESK AISDAQITAS SYFTNMFATW 1701SPSKARLHLQ GRSNAWRPQV NNPKEWLQVD FQKTMKVTGV TTQGVKSLLT 1751SMYVKEFLIS SSQDGHQWTL FFQNGKVKVF QGNQDSFTPV VNSLDPPLLT 1801RYLRIHPQSW VHQIALRMEV LGCEAQDLYD KTHTCPPCPA PELLGGPSVF 1851LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 1901REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG 1951QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY 2001KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL 2051 SLSPGK*pSYN FVIII 268 protein sequence (FVIII Fc with 144 AE-XTEN at aminoacid 18) SEQ ID NO: 114    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG   51PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG  101TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS  151ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL GELPVDARFP  201PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY  251DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG  301GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE  351GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM  401HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH  451RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE  501EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT  551WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY  601TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT  651DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR  701YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE  751NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL  801HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS  851MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL  901SKNNAIEPRS FSQNPPVLKR HQAEITRTTL QSDQEEIDYD DTISVEMKKE  951DFDIYDEDEN QSPRSFQKKT RHYFIAAVER LWDYGMSSSP HVLRNRAQSG 1001SVPQFKKVVF QEFTDGSFTQ PLYRGELNEH LGLLGPYIRA EVEDNIMVTF 1051RNQASRPYSF YSSLISYEED QRQGAEPRKN FVKPNETKTY FWKVQHHMAP 1101TKDEFDCKAW AYFSDVDLEK DVHSGLIGPL LVCHTNTLNP AHGRQVTVQE 1151FALFFTIFDE TKSWYFTENM ERNCRAPCNI QMEDPTFKEN YRFHAINGYI 1201MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV RKKEEYKMAL 1251YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL VYSNKCQTPL 1301GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL 1351LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV 1401FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS 1451MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN 1501PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF 1551QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG 1601CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 1651VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 1701LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 1751SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 1801KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*pSYN FVIII 269 protein sequence (FVIII Fc with 72 AE-XTEN at aminoacid 18) SEQ ID NO: 115    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG   51PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG  101TSTEPSEGSA PGASSSDLGE LPVDARFPPR VPKSFPFNTS VVYKKTLFVE  151FTDHLFNIAK PRPPWMGLLG PTIQAEVYDT VVITLKNMAS HPVSLHAVGV  201SYWKASEGAE YDDQTSQREK EDDKVFPGGS HTYVWQVLKE NGPMASDPLC  251LTYSYLSHVD LVKDLNSGLI GALLVCREGS LAKEKTQTLH KFILLFAVFD  301EGKSWHSETK NSLMQDRDAA SARAWPKMHT VNGYVNRSLP GLIGCHRKSV  351YWHVIGMGTT PEVHSIFLEG HTFLVRNHRQ ASLEISPITF LTAQTLLMDL  401GQFLLFCHIS SHQHDGMEAY VKVDSCPEEP QLRMKNNEEA EDYDDDLTDS  451EMDVVRFDDD NSPSFIQIRS VAKKHPKTWV HYIAAEEEDW DYAPLVLAPD  501DRSYKSQYLN NGPQRIGRKY KKVRFMAYTD ETFKTREAIQ HESGILGPLL  551YGEVGDTLLI IFKNQASRPY NIYPHGITDV RPLYSRRLPK GVKHLKDFPI  601LPGEIFKYKW TVTVEDGPTK SDPRCLTRYY SSFVNMERDL ASGLIGPLLI  651CYKESVDQRG NQIMSDKRNV ILFSVFDENR SWYLTENIQR FLPNPAGVQL  701EDPEFQASNI MHSINGYVFD SLQLSVCLHE VAYWYILSIG AQTDFLSVFF  751SGYTFKHKMV YEDTLTLFPF SGETVFMSME NPGLWILGCH NSDFRNRGMT  801ALLKVSSCDK NTGDYYEDSY EDISAYLLSK NNAIEPRSFS QNPPVLKRHQ  851AEITRTTLQS DQEEIDYDDT ISVEMKKEDF DIYDEDENQS PRSFQKKTRH  901YFIAAVERLW DYGMSSSPHV LRNRAQSGSV PQFKKVVFQE FTDGSFTQPL  951YRGELNEHLG LLGPYIRAEV EDNIMVTFRN QASRPYSFYS SLISYEEDQR 1001QGAEPRKNFV KPNETKTYFW KVQHHMAPTK DEFDCKAWAY FSDVDLEKDV 1051HSGLIGPLLV CHTNTLNPAH GRQVTVQEFA LFFTIFDETK SWYFTENMER 1101NCRAPCNIQM EDPTFKENYR FHAINGYIMD TLPGLVMAQD QRIRWYLLSM 1151GSNENIHSIH FSGHVFTVRK KEEYKMALYN LYPGVFETVE MLPSKAGIWR 1201VECLIGEHLH AGMSTLFLVY SNKCQTPLGM ASGHIRDFQI TASGQYGQWA 1251PKLARLHYSG SINAWSTKEP FSWIKVDLLA PMIIHGIKTQ GARQKFSSLY 1301ISQFIIMYSL DGKKWQTYRG NSTGTLMVFF GNVDSSGIKH NIFNPPIIAR 1351YIRLHPTHYS IRSTLRMELM GCDLNSCSMP LGMESKAISD AQITASSYFT 1401NMFATWSPSK ARLHLQGRSN AWRPQVNNPK EWLQVDFQKT MKVTGVTTQG 1451VKSLLTSMYV KEFLISSSQD GHQWTLFFQN GKVKVFQGNQ DSFTPVVNSL 1501DPPLLTRYLR IHPQSWVHQI ALRMEVLGCE AQDLYDKTHT CPPCPAPELL 1551GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH 1601NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT 1651ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG 1701QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH 1751YTQKSLSLSP GK*pSYNFVIII 271 protein sequence (FVIII Fc with 42 AE-XTEN at aminoacid 18) SEQ ID NO: 116    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP GSPAGSPTST   51EEGTSESATP ESGPGSEPAT SGSETPASSS DLGELPVDAR FPPRVPKSFP  101FNTSVVYKKT LFVEFTDHLF NIAKPRPPWM GLLGPTIQAE VYDTVVITLK  151NMASHPVSLH AVGVSYWKAS EGAEYDDQTS QREKEDDKVF PGGSHTYVWQ  201VLKENGPMAS DPLCLTYSYL SHVDLVKDLN SGLIGALLVC REGSLAKEKT  251QTLHKFILLF AVFDEGKSWH SETKNSLMQD RDAASARAWP KMHTVNGYVN  301RSLPGLIGCH RKSVYWHVIG MGTTPEVHSI FLEGHTFLVR NHRQASLEIS  351PITFLTAQTL LMDLGQFLLF CHISSHQHDG MEAYVKVDSC PEEPQLRMKN  401NEEAEDYDDD LTDSEMDVVR FDDDNSPSFI QIRSVAKKHP KTWVHYIAAE  451EEDWDYAPLV LAPDDRSYKS QYLNNGPQRI GRKYKKVRFM AYTDETFKTR  501EAIQHESGIL GPLLYGEVGD TLLIIFKNQA SRPYNIYPHG ITDVRPLYSR  551RLPKGVKHLK DFPILPGEIF KYKWTVTVED GPTKSDPRCL TRYYSSFVNM  601ERDLASGLIG PLLICYKESV DQRGNQIMSD KRNVILFSVF DENRSWYLTE  651NIQRFLPNPA GVQLEDPEFQ ASNIMHSING YVFDSLQLSV CLHEVAYWYI  701LSIGAQTDFL SVFFSGYTFK HKMVYEDTLT LFPFSGETVF MSMENPGLWI  751LGCHNSDFRN RGMTALLKVS SCDKNTGDYY EDSYEDISAY LLSKNNAIEP  801RSFSQNPPVL KRHQAEITRT TLQSDQEEID YDDTISVEMK KEDFDIYDED  851ENQSPRSFQK KTRHYFIAAV ERLWDYGMSS SPHVLRNRAQ SGSVPQFKKV  901VFQEFTDGSF TQPLYRGELN EHLGLLGPYI RAEVEDNIMV TFRNQASRPY  951SFYSSLISYE EDQRQGAEPR KNFVKPNETK TYFWKVQHHM APTKDEFDCK 1001AWAYFSDVDL EKDVHSGLIG PLLVCHTNTL NPAHGRQVTV QEFALFFTIF 1051DETKSWYFTE NMERNCRAPC NIQMEDPTFK ENYRFHAING YIMDTLPGLV 1101MAQDQRIRWY LLSMGSNENI HSIHFSGHVF TVRKKEEYKM ALYNLYPGVF 1151ETVEMLPSKA GIWRVECLIG EHLHAGMSTL FLVYSNKCQT PLGMASGHIR 1201DFQITASGQY GQWAPKLARL HYSGSINAWS TKEPFSWIKV DLLAPMIIHG 1251IKTQGARQKF SSLYISQFII MYSLDGKKWQ TYRGNSTGTL MVFFGNVDSS 1301GIKHNIFNPP IIARYIRLHP THYSIRSTLR MELMGCDLNS CSMPLGMESK 1351AISDAQITAS SYFTNMFATW SPSKARLHLQ GRSNAWRPQV NNPKEWLQVD 1401FQKTMKVTGV TTQGVKSLLT SMYVKEFLIS SSQDGHQWTL FFQNGKVKVF 1451QGNQDSFTPV VNSLDPPLLT RYLRIHPQSW VHQIALRMEV LGCEAQDLYD 1501KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP 1551EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC 1601KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG 1651FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN 1701VFSCSVMHEA LHNHYTQKSL SLSPGK*pSYN FVIII protein sequence 272 ( FVIII with 144 AE XTEN at aminoacid 18 and 244 AE XTEN in B-domain-no Fc) SEQ ID NO: 117    1MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG   51PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG  101TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS  151ESATPESGPG SPAGSPTSTE EGSPAGSPTS TEEGASSSDL GELPVDARFP  201PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY  251DTVVITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG  301GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE  351GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM  401HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH  451RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE  501EPQLRMKNNE EAEDYDDDLT DSEMDVVRFD DDNSPSFIQI RSVAKKHPKT  551WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY  601TDETFKTREA IQHESGILGP LLYGEVGDTL LIIFKNQASR PYNIYPHGIT  651DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR  701YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE  751NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL  801HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS  851MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDISAYLL  901SKNNAIEPRS FSQNGAPGTS ESATPESGPG SEPATSGSET PGTSESATPE  951SGPGSEPATS GSETPGTSES ATPESGPGTS TEPSEGSAPG SPAGSPTSTE 1001EGTSESATPE SGPGSEPATS GSETPGTSES ATPESGPGSP AGSPTSTEEG 1051SPAGSPTSTE EGTSTEPSEG SAPGTSESAT PESGPGTSES ATPESGPGTS 1101ESATPESGPG SEPATSGSET PGSEPATSGS ETPGSPAGSP TSTEEGTSTE 1151PSEGSAPGTS TEPSEGSAPG SEPATSGSET PGTSESATPE SGPGTSTEPS 1201EGSAPASSPP VLKRHQAEIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD 1251EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK 1301KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR 1351PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD 1401CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT 1451IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG 1501LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG 1551VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH 1601IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII 1651HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD 1701SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME 1751SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ 1801VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK 1851VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL 1901 Y*pSYN VWF 031 protein sequence (VWF D1D2D′D3-48 aa long thrombincleavable GS linker-Fc) SEQ ID NO: 118    1MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM   51YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG  101TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL  151SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC  201ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC  251EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME  301YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC  351VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD  401NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG  451LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM  501DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG  551NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS  601PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL  651NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD  701CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD  751AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM  801SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV  851CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS  901NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE  951THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201VAGRRFASGK KVILNPSDPE HCQICHCDVV NLTCEACQEP ISGGGGSGGG 1251GSGGGGSGGG GSGGGGSGGG GSLVPRGSGG GGSGGGGSDK THTCPPCPAP 1301ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV 1351EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI 1401EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE 1451SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL 1501HNHYTQKSLS LSPGK*pSYN VWF 034 protein sequence (VWF D1D2D′D3-288AE XTEN-35 aalong thrombin cleavable GS linker-Fc) SEQ ID NO: 119    1MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM   51YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG  101TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL  151SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC  201ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC  251EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME  301YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC  351VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD  401NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG  451LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM  501DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG  551NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS  601PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL  651NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD  701CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD  751AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM  801SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV  851CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS  901NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE  951THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP ISGTSESATP 1251ESGPGSEPAT SGSETPGTSE SATPESGPGS EPATSGSETP GTSESATPES 1301GPGTSTEPSE GSAPGSPAGS PTSTEEGTSE SATPESGPGS EPATSGSETP 1351GTSESATPES GPGSPAGSPT STEEGSPAGS PTSTEEGTST EPSEGSAPGT 1401SESATPESGP GTSESATPES GPGTSESATP ESGPGSEPAT SGSETPGSEP 1451ATSGSETPGS PAGSPTSTEE GTSTEPSEGS APGTSTEPSE GSAPGSEPAT 1501SGSETPGTSE SATPESGPGT STEPSEGSAP DIGGGGGSGG GGSLVPRGSG 1551GDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE 1601DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 1651KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV 1701KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 1751GNVFSCSVMH EALHNHYTQK SLSLSPGK*pSYN VWF 036 protein sequence (VWF D1D2D′D-98 aa long thrombincleavable GS linker-Fc) SEQ ID NO: 120    1MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM   51YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG  101TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL  151SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC  201ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC  251EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME  301YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC  351VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD  401NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG  451LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM  501DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG  551NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS  601PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL  651NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD  701CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD  751AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM  801SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV  851CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS  901NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE  951THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD 1001GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI 1051MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF 1101CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA 1151PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP ISGGGGSGGG 1251GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG GSGGGGSGGG 1301GSGGGGSGGG GSGGGGSGGG GSLVPRGSGG GGSGGGGSDK THTCPPCPAP 1351ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV 1401EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI 1451EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE 1501SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL 1551HNHYTQKSLS LSPGK*pSYN Fc-015 protein sequence (IgG-Fc domain) SEQ ID NO: 121    1METDTLLLWV LLLWVPGSTG DKTHTCPPCP APELLGGPSV FLFPPKPKDT   51LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY  101RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT  151LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS  201DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK*

Example 15: FVIII-XTEN-Fc:VWF-Fc Heterodimers have Maintained NormalFVIII Specific Activity as Compared to Wild Type BDD-FVIII

The FVIII specific activity of FVIII-XTEN-Fc:VWF-Fc heterodimers weredetermined. Heterodimers were purified using a two-step chromatographyprocess. A weak anion exchange resin was used, followed by affinitychromatography. The final purified product had acceptable purity bySEC-HPLC. The specific activity was compared to B-domain deleted FVIII(BDD-FVIII), as measured by FVIII chromogenic assay and A280concentration. The data are presented in Table 26. All tested moleculeshad demonstrated comparable FVIII specific activities to BDD-FVIII.Purity and the presence of each moiety of the molecules were confirmedby SDS-PAGE and western blotting.

TABLE 26 FVIII specific activity of FVIII-XTEN-Fc:VWF-Fc heterodimersFVIII FVIII FVIII FVIII FVIII FVIII 207 FVIII-66 155/ 155/ 169/ 205/169/ Construct scBDDFVIII dcBDDFVIII) vWF31 vWF39 vWF31 vWF31 vWF34Measured 1473 1592 1534 1796 1511 1345 1505 Specific Activity (IU/nmol)

The half-lives of rFVIII-XTEN/D′D3 and BDD-FVIII were compared in HemAMice (FIG. 15; Table 27). As FIG. 15 shows, rFVIII-XTEN/D′D3 achieved ahalf-life that was four fold longer than the half-life achieved byBDD-FVIII.

TABLE 27 rFVIII-XTEN/D′D3 and BDD-FVIII in HemA mice 5 minutes Cl AUC_DRecovery HL MRT (mL/hr/ Vss (hr*kg* Treatment (%) (hr) (hr) kg) (mL/kg)mIU/mL/mIU) BDD-FVIII 89 7.6 11 4.5 49.2 0.22 rFVIIIFc 78 16 20 2.9 57.80.35 rFVIII- 86 30 36 1.8 63.4 0.57 XTEN/D′D3

Example 16: FVIII-XTEN-Fc:VWF-Fc Heterodimer's Potency (FVIII Activity)in Hemostasis as Measured by One Stage aPTT Assay

The potency of FVIII-XTEN-Fc:VWF-Fc heterodimers in hemostasis wasevaluated by their FVIII specific aPTT activity as summarized in Table28. As demonstrated by Table 28, while the addition of the VWF D′D3fragment and the insertion of XTEN into the intra-domains of FVIIIreduce the FVIII specific aPTT activity of the heterodimers (asindicated by the FVIII155NWF031 data and the FVIII205NWF031 data), XTENinsertions in the FVIII B domain region or C-terminus of the VWF D′D3fragment have no negative effect on the FVIII specific aPTT activity (asindicated by the FVIII169NWF031 data and the FVIII169NWF034 data).Compared to dual-chain BDD-FVIII (dcBDD-FVIII), FVIII155NWF031,FVIII169NWF031, FVIII169NWF034 and VWF205NWF031 showed reduction ofspecific aPTT activity by 2.5-fold, 2.8-fold, 2.6-fold and 5.5-fold,respectively.

TABLE 28 FVIII specific aPTT activity of FVIII-XTEN-Fc:VWF-Fcheterodimers FVIII 207 FVIII-66 FVIII FVIII FVIII FVIII scBDD- dcBDD-155/ 169/ 205/ 169/ Construct FVIII FVIII VWF31 VWF31 VWF31 VWF34Measured 818 ± 1188 ± 448 ± 416 ± 214 ± 436 ± Specific 153 213 111 70 38189 aPTT Activity (IU/nmol)FVIII Specific aPTT Assay

FVIII variants were diluted with aPTT buffer (0.15 M NaCl, 0.05 MTris-HCl, 1% BSA, pH 7.4) to the linear assay range (200-1.6 mU/mL). 50μL of diluted samples or standards were sequentially mixed with 50 μL of37° C. naïve human HemA pooled plasma, 50 μL of 37° C. aPTT reagent(ACTIN® FSL activated cephaloplastin reagent—Dade Behring, reference#B4219-2) and incubated at 37° C. for 4 minutes. 50 μl of 20 mM CaCl₂)(Dade Behring [reference #ORFO37]) was then added to the reactionmixture to start the clotting reactions. Using the clotting time of eachsample (the length of time from the addition of CaCl₂) until the onsetof clot formation), the aPTT activity was calculated against thestandard that was generated with the 8^(th) international standard FVIIIconcentrate. Specific aPTT activity was calculated against the proteinconcentration of each molecule that measured by OD280.

Example 17: In Vivo Efficacy of FVIII-XTEN-Fc:VWF-Fc Heterodimer in HemAMice Tail Clip Bleeding Model

To further access the hemostasis potency of the heterodimers, the acuteefficacy of FVIII169NWF034 and FVIII205NWF031 was evaluated incomparison with BDD-FVIII in the HemA mice Tail clip bleeding model.HemA mice were treated with a single IV injection of BDD-FVIII at 200,65 and 20 IU/kg to generate the post tail clip injury blood loss controllevel. Blood loss from mice treated with 200 IU/kg of FVIII169NWF034 orFVIII205NWF031 was compared to that of the BDD-FVIII treated controlgroup mice to estimate their potency on hemostasis. Vehicle treatedanimals were used to generate blood loss baseline for the model. Asshown in FIG. 16, significant reduction in blood loss was observed fromall FVIII treatment groups compared to that of the vehicle treatedanimals (p<0.05). Both FVIII169NWF034 and FVIII205NWF031 are efficaciousin the HemA mice Tail Clip model. Compared to BDD-FVIII, about 3 foldlower potency was observed for FVIII169NWF034, as demonstrated by thesimilar blood loss reduction achieved by 65 IU/kg BDD-FVIII and 200IU/kg FVIII169NWF034. As for FVIII205NWF034, a 10 fold potency reductionhas been observed, as demonstrated by the similar blood loss reductionachieved by 20 IU/kg BDD-FVIII and 200 IU/kg FVIII205NWF031.

Even though FVII169NWF034 and FVIII205NWF031 had similar specific FVIIIchromogenic activity compared to rBDD-FVIII, their FVIII aPTT activityand in vivo potency were both reduced due to the modifications of themolecules. Those data indicate that the aPTT activity of a FVIIImolecule is a more accurate measurement on predicating its in vivopotency on hemostasis than the FVIII chromogenic activity.

HemA Mice Tail Clip Bleeding Model

8-10 weeks old male HemA mice were used for the study. Prior to tailclip injury, mice were anesthetized with a 50 mg/kg Ketamine/0.5 mg/kgDexmedetomidine cocktail and placed on a 37° C. heating pad to helpmaintain the body temperature. The tails of the mice were then beimmersed in 37° C. water for 10 minutes to dilate the lateral vein.After vein dilation, rFVIII or vehicle solution were injected via thetail vein and 5 min later, the distal 1 cm of the tail was cut off usinga #11 scalpel with straight edge. The shed blood was collected into 13ml of 37° C. warm saline for 30 minutes and the mice were theneuthanized while still under anesthesia by bilateral thoracotomy. Bloodloss was quantified gravimetrically by weight change of the bloodcollection tubes before and after blood was collected in gram, whichtranslated into milliliter (mL) of blood loss volume (1 g weightchange=1 mL blood loss).

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

All patents and publications cited herein are incorporated by referenceherein in their entirety.

1. A chimeric protein comprising (i) a von Willebrand Factor (VWF)protein comprising a D′ domain and a D3 domain of VWF, (ii) an XTENsequence, and (iii) a FVIII protein, wherein the VWF fragment and theXTEN sequence are linked by an optional linker, wherein the VWF fragmentor the XTEN sequence is linked to or associated with the FVIII protein.2. A chimeric protein comprising a formula, which comprises:V-X-FVIII,  (a)FVIII-X-V,  (b)V-X:FVIII,  (c)X-V:FVIII,  (d)FVIII:V-X, or  (e)FVIII:X-V,  (f) wherein V comprises a VWF fragment, X comprises one ormore XTEN sequences, FVIII comprises a FVIII protein; (−) is a peptidebond or one or more amino acids; and (:) is a covalent bond or anon-covalent bond.
 3. The chimeric protein of claim 1, wherein the XTENsequence is linked to the FVIII protein by a linker. 4-12. (canceled)13. The chimeric protein of claim 1, further comprising a first Igconstant region or a portion thereof. 14-16. (canceled)
 17. The chimericprotein of claim 13, wherein the chimeric protein comprises a second Igconstant region, wherein the second Ig constant region is linked to theVWF fragment by a linker. 18-19. (canceled)
 20. The chimeric protein ofclaim 17, wherein the second Ig constant region or a portion thereof isassociated with the first Ig constant region or a portion thereof. 21.The chimeric protein of claim 17, wherein the second Ig constant regionor a portion thereof is associated with the first Ig constant region ora portion thereof by a covalent bond. 22-67. (canceled)
 68. The chimericprotein of claim 1, wherein the FVIII protein is linked to an XTENsequence at the C-terminus or the N-terminus of the FVIII protein orinserted immediately downstream of one or more amino acids in the FVIIIprotein or any combinations thereof.
 69. (canceled)
 70. The chimericprotein of claim 1, wherein the FVIII protein comprises one or moredomains of FVIII selected from the group consisting of an A1 domain, a1acidic region, an A2 domain, a2 acidic region, a B domain, an A3 domain,a3 acidic region, a C1 domain, a C2 domain, one or more fragmentsthereof, and any combinations thereof. 71-74. (canceled)
 75. Thechimeric protein of claim 1, wherein the FVIII protein comprises a Bdomain or a portion thereof. 76-89. (canceled)
 90. The chimeric proteinof claim 1, wherein the amino acid sequence of the D′ domain is at least90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to866 of SEQ ID NO:
 2. 91. The chimeric protein of claim 1, wherein theamino acid sequence of the D3 domain is at least 90%, 95%, 96%, 97%,98%, or 99%, identical to amino acids 867 to 1240 of SEQ ID NO: 2.92-95. (canceled)
 96. The chimeric protein of claim 91, wherein the VWFfragment contains at least one amino acid substitution at a residuecorresponding to residue 1099, residue 1142, or both residues 1099 and1142 of SEQ ID NO:
 2. 97-119. (canceled)
 120. A polynucleotide or a setof polynucleotides encoding the chimeric protein of claim
 1. 121.(canceled)
 122. A vector comprising the polynucleotide or set ofpolynucleotides of claim 120 and one or more promoter operably linked tothe polynucleotide or the set of polynucleotides.
 123. (canceled)
 124. Ahost cell comprising the polynucleotide of or set of polynucleotides ofclaim
 120. 125-126. (canceled)
 127. A pharmaceutical compositioncomprising the chimeric protein of claim 1, and a pharmaceuticallyacceptable carrier. 128-137. (canceled)
 138. A method of extending orincreasing half-life of a FVIII protein in a subject, wherein the methodcomprises administering an effective amount of the chimeric protein ofclaim 1 to a subject in need thereof, wherein the VWF fragment binds tothe FVIII protein and thus extends or increases half-life of the FVIIIprotein. 139-142. (canceled)
 143. A method of treating a bleedingdisease or disorder in a subject in need thereof comprisingadministering an effective amount of the chimeric protein of claim 1,wherein the bleeding disease or disorder is selected from the groupconsisting of a bleeding coagulation disorder, hemarthrosis, musclebleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage,trauma, trauma capitis, gastrointestinal bleeding, intracranialhemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bonefracture, central nervous system bleeding, bleeding in theretropharyngeal space, bleeding in the retroperitoneal space, andbleeding in the iliopsoas sheath. 144-147. (canceled)
 148. A method ofmaking a chimeric protein, comprising transfecting a host cell with thepolynucleotide or set of polynucleotides of claim 120 and expressing thechimeric protein in the host cell. 149-165. (canceled)